Antibodies specific for urokinase-type plasminogen activator and methods of use thereof

ABSTRACT

The present disclosure relates to antibodies specific for urokinase-type plasminogen activator (uPA). According to certain embodiments, the anti-uPA antibody specifically binds to the active form of uPA. In certain aspects, the anti-uPA antibody that specifically binds to active uPA binds specifically to the active form of human uPA (e.g., the antibody does not cross-react with active forms of uPA from non-human organisms). In certain aspects, an anti-uPA antibody of the present disclosure competes for specific binding to uPA with plasminogen activator inhibitor type 1 (PAI-1), where binding of the antibody to uPA results in internalization of a complex that includes the antibody, uPA, and urokinase-type plasminogen activator receptor (uPAR). Also provided are antibodies that specifically bind to uPA and compete for binding to uPA with a synthetic ligand of uPA. The disclosure also provides anti-uPA antibody conjugates and compositions (e.g., pharmaceutical formulations) comprising the antibodies and/or conjugates. Methods and kits related to the anti-uPA antibodies, conjugates, or formulations including the antibodies and/or conjugates are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/162,439, filed Jan. 23, 2014, now U.S. Pat. No. 9,255,155, issuedFeb. 9, 2016, which application claims priority benefit of U.S.provisional application Ser. No. 61/759,321, filed Jan. 31, 2013, whichapplication is incorporated herein by reference in its entirety.

INTRODUCTION

Certain diseases have been found to be dependent upon misregulatedenzyme function, including proteases. In particular, proteases have beenimplicated in a number of functions essential for cancer progression.These include extracellular matrix remodeling, release of cytokines, andloss of apoptotic response. One particular protease that has beenimplicated in cancer progression is the serine protease urokinase typeplasminogen activator (uPA) (Ahmad et al. (2009) J. Cell. Biochem107(3):516-27; Ulisse et al. (2009) Curr. Cancer Drug Targets 9:32-71).Single chain pro-uPA is secreted as a 411 amino acid zymogen and becomesactivated by plasmin cleavage of the K158-I159 peptide bridge, thusgenerating uPA, a two-chain molecule held together by a single disulfidebond at C148-C279. A further cleavage releases an amino terminal domainthat includes an “EGF-like” and a “kringle” domain. The remainingcarboxyterminal region (low molecular weight uPA) retains full catalyticactivity. Active uPA activates plasminogen to plasmin, a broad spectrumserine protease. When uPA is bound to uPAR, the uPA-uPAR complex is notinternalized and remains on the cell surface. When receptor-bound uPA isbound by the specific plasminogen activator inhibitor type 1 (PAI-1),the resulting PAI-1-uPA-uPAR complex is internalized. Uponinternalization, uPA and PAI-1 are degraded in the lysosomes, while uPARis recycled back to the cell surface.

SUMMARY

The present disclosure relates to antibodies specific for urokinase-typeplasminogen activator (uPA). According to certain embodiments, theanti-uPA antibody specifically binds to the active form of uPA. Incertain aspects, the anti-uPA antibody that specifically binds to activeuPA binds specifically to the active form of human uPA (e.g., theantibody does not cross-react with active forms of uPA from non-humanorganisms). In certain aspects, an anti-uPA antibody of the presentdisclosure competes for specific binding to uPA with plasminogenactivator inhibitor type 1 (PAI-1), where binding of the antibody to uPAresults in internalization of a complex that includes the antibody, uPA,and urokinase-type plasminogen activator receptor (uPAR). Also providedare antibodies that specifically bind to uPA and compete for binding touPA with a synthetic ligand of uPA. The disclosure also providesanti-uPA antibody conjugates and compositions (e.g., pharmaceuticalformulations) comprising the antibodies and/or conjugates. Methods andkits related to the anti-uPA antibodies, conjugates, or formulationsincluding the antibodies and/or conjugates are also provided.

Accordingly, the present disclosure provides antibodies thatspecifically bind to urokinase-type plasminogen activator (uPA). Theantibodies may be non-human antibodies (e.g., mouse antibodies, rabbitantibodies, or any non-human antibody), humanized antibodies, or fullyhuman antibodies (e.g., a fully human recombinant antibody identifiedfrom a phage display library, or the like). Antibodies of the presentdisclosure bind to one or more epitopes of uPA. According to certainembodiments, the antibody binds to the heavy (B) chain of uPA. Such anantibody may bind to the serine protease domain (SPD) of uPA. Anantibody that binds to the SPD of uPA may bind to the active site ofuPA, or may bind to a region outside of the active site. In certainaspects, the anti-uPA antibody does not bind to the autolysis loop ofuPA. According to certain embodiments, the anti-uPA antibody does notbind to the activation domain of uPA. In certain aspects, the antibodyspecifically binds to urokinase-type plasminogen activator (uPA) andcompetes for binding to uPA with a ligand of uPA, e.g., a syntheticligand of uPA. In certain aspects, the antibody competes for binding touPA with a uPA ligand, which ligand binds to the active site of uPAwithout binding to an exosite of uPA (e.g., the ligand binds to theactive site of uPA independent of interactions with an exosite (e.g., aregion outside of the active site) of uPA).

The antibodies provided by the present disclosure may include one ormore (e.g., 1, 2, 3, 4, 5, or all 6) complementary determining regions(CDRs) set forth in Table 1, FIG. 11 and SEQ ID NOs: 1-6, orconservative variants of one or more such CDRs.

According to certain embodiments, an antibody of the present disclosurecompetes for specific binding to uPA with plasminogen activatorinhibitor type 1 (PAI-1). Binding of the antibody to uPA results ininternalization of a complex that includes the antibody, uPA, andurokinase-type plasminogen activator receptor (uPAR). In certainaspects, the complex does not include PAI-1.

In certain aspects, provided is an isolated antibody that specificallybinds to uPA (e.g., specifically binds to the active form of uPA (e.g.,two-chain high molecular weight uPA and/or two-chain low molecularweight uPA)), where the antibody competes for binding to uPA with asynthetic ligand (e.g., a small molecule such as p-aminobenzamidine, asynthetic peptide ligand, and the like) of uPA. According to certainembodiments, the substrate is a synthetic peptide ligand, which ligandis EGR-CMK (Glu-Gly-Arg-CMK). In other aspects, the ligand is thesynthetic peptide ligand S-2444 (H-D-Glu-Gly-Arg-p-nitroanilide).

Any antibody described above may be an antibody that competes forspecific binding to uPA with an antibody that includes: a heavy chaincomplementary determining region 1 (HCDR1) having the amino acidsequence of SEQ ID NO:1; a heavy chain complementary determining region2 (HCDR2) having the amino acid sequence of SEQ ID NO:2; a heavy chaincomplementary determining region 3 (HCDR3) having the amino acidsequence of SEQ ID NO:3; a light chain complementary determining region1 (LCDR1) having the amino acid sequence of SEQ ID NO:4; a light chaincomplementary determining region 2 (LCDR2) having the amino acidsequence of SEQ ID NO:5; and a light chain complementary determiningregion 3 (LCDR3) having the amino acid sequence of SEQ ID NO:6. Incertain aspects, such an antibody includes: a heavy chain complementarydetermining region 1 (HCDR1) having the amino acid sequence of SEQ IDNO:1; a heavy chain complementary determining region 2 (HCDR2) havingthe amino acid sequence of SEQ ID NO:2; a heavy chain complementarydetermining region 3 (HCDR3) having the amino acid sequence of SEQ IDNO:3; a light chain complementary determining region 1 (LCDR1) havingthe amino acid sequence of SEQ ID NO:4; a light chain complementarydetermining region 2 (LCDR2) having the amino acid sequence of SEQ IDNO:5; and a light chain complementary determining region 3 (LCDR3)having the amino acid sequence of SEQ ID NO:6. Optionally, the antibodyincludes a heavy chain polypeptide that includes a variable regionincluding the amino acid sequence set forth in SEQ ID NO:7, a lightchain polypeptide that includes a variable region including the aminoacid sequence set forth in SEQ ID NO:8, or heavy and light chainpolypeptides that include variable regions including the amino acidsequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.

Also provided is an isolated antibody that includes: a heavy chaincomplementary determining region 1 (HCDR1) having the amino acidsequence of SEQ ID NO:1; a heavy chain complementary determining region2 (HCDR2) having the amino acid sequence of SEQ ID NO:2; a heavy chaincomplementary determining region 3 (HCDR3) having the amino acidsequence of SEQ ID NO:3; a light chain complementary determining region1 (LCDR1) having the amino acid sequence of SEQ ID NO:4; a light chaincomplementary determining region 2 (LCDR2) having the amino acidsequence of SEQ ID NO:5; and a light chain complementary determiningregion 3 (LCDR3) having the amino acid sequence of SEQ ID NO:6.Optionally, the antibody includes a heavy chain polypeptide thatincludes a variable region including the amino acid sequence set forthin SEQ ID NO:7, a light chain polypeptide that includes a variableregion including the amino acid sequence set forth in SEQ ID NO:8, orheavy and light chain polypeptides that include variable regionsincluding the amino acid sequences set forth in SEQ ID NO:7 and SEQ IDNO:8, respectively.

Any antibody described above may, upon binding to uPA, result ininternalization of a complex comprising the antibody, uPA, andurokinase-type plasminogen activator receptor (uPAR). According tocertain embodiments, the internalized antibody/uPA/uPAR complex does notinclude PAI-1.

Any of the antibodies described above may be a “full-length” or “whole”antibody (e.g., a full-length IgG antibody), or may be an antibodyfragment. The the antibody is an antibody fragment, the fragment may bean Fv, scFv, Fab, F(ab′)2, Fab′, or any other type of antibody fragmentof interest.

Nucleic acids encoding any of the antibodies of the present inventionare also provided. For example, the nuceic acid sequences encoding theU33 heavy and light chain variable polypeptides are provided in FIG. 11,panel B, the nucleic acids may be present in an expression vector forproduction of any antibody of the present disclosure in a cell.

Antibody conjugates are also provided. The conjugates include anyantibody of the present disclosure and an agent. The agent may beselected from a therapeutic agent, and a labeling agent, or an agentuseful for both therapeutic and labeling purposes. When the conjugateincludes a therapeutic agent, the agent may be a cytotoxic agent, suchas a radionuclide, a chemotherapeutic agent, a toxin, or any othertherapeutic agent of interest. When the conjugate includes a labelingagent, the agent may be an in vivo imaging agent. Such agents find usein, e.g., the detection, diagnosis and/or prognosis of uPA relateddiseases (e.g., cancer). Such agents find use in in vivo imagingapplications such as near-infrared (NIR) imaging, single photon emissioncomputed tomography (SPECT), and/or the like, for detection, diagnosisand/or prognosis of uPA related diseases (e.g., cancer).

Also provided are sterile pharmaceutical compositions that include anyof the antibodies of the present disclosure described herein (or any ofthe antibody conjugates of the present disclosure described herein), anda pharmaceutically acceptable carrier.

In certain aspects, the present disclosure provides methods of treatinguPA-related diseases. For example, according to one embodiment, providedis a method for treating cancer. The method includes the step ofadministering to a subject in need thereof a therapeutically effectiveamount of any anti-uPA antibodies of the present disclosure, anyanti-uPA antibody conjugate of the present disclosure, or anypharmaceutical composition that includes such an antibody or conjugate.When the treatment method is a method of treating cancer, the cancer maybe any cancer in which uPA activity is involved (e.g., by causing cellcycle dysregulation, facilitating metastasis and/or invasiveness of thecancer, and/or the like). In certain aspects, the method is a method fortreating a cancer selected from prostate cancer (e.g.,castration-resistant prostate cancer), breast cancer, gastric cancer,colorectal cancer, esophageal cancer, renal cancer, endometrial cancer,ovarian cancer, and combinations thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1, panels A-G, depict uPA expression in prostate cancer cell linesand prostate cancer tissue microarray sections. Panel A shows mRNAlevels of uPAR (top) and uPA and PAI-1 (bottom) analyzed usingquantitative RT-PCR in prostate cancer cell lines and normal humanprostate epithelial cells. Panel B shows qRT-PCR analysis of the PAScomponents in PC3 and DU145 cells cultured under 5% O₂ for 72 hrs. Theincrease in mRNA expression is compared to cells grown under normoxia.The left bar, middle bar, and right bar for the PC3 and DU145 cellexperiments correspond to uPAR, uPA and PAI-1, respectively. Panel Cshows IHC staining of a PC3 xenograft section (left) and a DU145xenograft section (right) for total uPA protein using the antibodysc-14019. Panels D-G show uPA protein in prostate cancer tissuemicroarray sections by immunofluorescence. H&E stained sections areviewed on the left with the merged fluorescence channels on the rightwith uPA (green), E-cadherin (red) and nuclei (DAPI). The sectionsstained are: adenocarcinoma, grade II, T1N0M0 (2+3=5) (Panel D);adenocarcinoma, grade II, T2N0M0 (1+4=5) (Panel E); adenocarcinoma,grade IV, T3bN1M0 (4+5=9) (Panel F); bone metastasis, grade IV (PanelG).

FIG. 2, panel A shows specific binding of the U33 Fab to the active formof uPA. Serial dilutions of pure U33 were added to uPA coated plates andincubated for 1 hour. The amount of Fab bound to uPA was determined byELISA. U33 Fab was detected only in wells coated with human active uPA.Panel B depicts inhibition of human uPA by U33. The proteolytic activityof human or mouse uPA was read in absence and presence of 1 μM of U33.The enzyme activity is expressed as percentage of the uPA activity inabsence of Fab (100%). Panel C shows inhibition of uPA bound to uPAR.Serial dilutions of U33-Fab were added to uPAR-uPA coated plates andincubated for 1 hour and the activity of uPA was read. Panel D depictsprevention of uPA binding to PAI-1 coated plates by U33. Serialdilutions of pure U33 (4 μM to 31.2 nM) were pre-incubated overnightwith uPA and added to PAI-1 coated plates. The amount of uPA bound toPAI was determined by ELISA. Panel E shows that U33 does not bind to uPAinhibited by a CMK inhibitor. Serial dilutions of U33 were added to auPA-coated plate pre-incubated with and without 1 μM CMK. The amount ofU33 Fab bound to uPA was determined by ELISA. Panel F shows aLineweaver-Burk plot demonstrating that U33 IgG is a competitiveinhibitor of uPA.

FIG. 3, panel A shows the specificity of U33-IgG for uPA compared to apanel of proteases. Proteases were treated with 1 μM of U33 IgG in thepresence of fluorogenic substrate. Panel B depicts inhibition oftrypsin-like proteolytic activity in the conditioned media of PC3 andDU145 cells by U33 IgG. Conditioned media were incubated with thetrypsin cleavable fluorogenic substrate Z-Gly-Gly-Arg-AMC (ex. 355 nm;em. 460 nm) at 400 μM in the presence and absence of U33 IgG. Panel Cshows mass spectrometry proteomic analysis of proteases in theconditioned media from PC3 and DU145 cells. Panel D depicts PC3 cellularinternalization of ¹¹¹In-U33 IgG at 37° C. and 4° C. in conditionedmedia. PC3 cells were incubated with 50 nM of radiolabeled antibody atthe indicated time points and were washed and treated with an acidicbuffer to remove non-covalently bound and non-internalized ¹¹¹In-U33IgG. Each time point was performed in triplicate. Panel E showsinternalization of ¹¹¹In-U33 IgG at the 120 min time point by the cellslines PC3, DU145 and CWR22Rv1 in conditioned media. Blocking wasperformed by adding 100 μg of cold U33 IgG to the media prior toaddition of radiolabeled antibody. ¹¹¹In-A11 IgG was used as the isotypecontrol antibody for the PC3 cells.

FIG. 4, panel A shows selectivity of U33 binding to uPA compared tothrombin, HGFA, hepsin, chymotrypsin, and trypsin. Panel B showsselectivity of U33 binding to uPA compared to MT-SP1 and plasmin. PanelC shows selectivity of U33 binding to uPA compared to tPA. Panel D showsselectivity of U33 binding to human uPA compared to mouse uPA.

FIG. 5, panel A shows U33 binding to uPA versus pro-uPA (zymogen uPA).Panel B shows U33 binding to uPA versis uPA bound to uPAR. Panel C showsU33 binding to uPA versus uPA inhibited by PAI-1.

FIG. 6, panel A shows U33 binding to uPA versus uPA inhibited by a CMKinhibitory peptide. Panel B shows displacement of p-aminobenzamidine byU33.

FIG. 7, panels A-D, show near-infrared (NIR) optical imaging of prostatecancer xenografts using AF680-U33 IgG. In panel A, mice bearing PC3,DU145 and CWR22Rv1 xenografts were tail-vein injected with 2 nmol ofAF680-U33 IgG and imaged using NIR optical imaging. The images shown arerepresentative of n=3 mice/xenograft and were acquired 72 hrspost-injection. Panel B shows the resected PC3 tumor at 72 hrsfluorescence intensity (left) and a tumor section demonstrating probepenetration and localization by fluorescence microscopy (right). Panel Cdepicts probe fluorescence intensity (left) and localization (right) inthe liver of a PC3 xenograft mouse. Panel D shows a graph depicting thelocalization of AF680-U33 IgG as fluorescence efficiency of the tumorROIs for the mice imaged using NIR optical imaging. Included in thegraph are the data for the mice imaged with the isotype controlAF680-A11 IgG in PC3 xenografts.

FIG. 8, panels A-C, depict SPECT/CT imaging and biodistribution of¹¹¹In-U33 IgG in xenograft bearing mice. Panel A shows SPECT imagingwith ¹¹¹In-U33 IgG in a PC3 xenograft model. Depicted are SPECT/CTimages shown as a three-dimensional volume rendering of the SPECT data(blue) overlaid onto surface rendered CT data and a reconstructedtransverse view using a rainbow color scale to show uptake (below).Imaging is representative of n=3 mice imaged with ¹¹¹In-U33 IgG at 72hrs post-injection. Each animal for imaging received 2.5 μg of antibodycorresponding to 220 μCi of activity. As shown in panel B, probebiodistribution was determined by radioactivity assays in PC3 tumorbearing mice (n=4 for each time point). Tissues were harvested at 24, 48and 72 hrs after injection of ¹¹¹In-U33 IgG (25 Probe uptake is reportedas percent injected dose per gram (% ID/g). For each tissue type, theleft bar, middle bar, and right bar correspond to 24 hrs, 48 hrs and 72hrs, respectively. Panel C depicts tumor uptake specificity measured at72 hrs post-injection (n=4 mice for each treatment). PC3 xenograftbearing mice were treated with isotype control ¹¹¹In-A11 IgG (25 μCi)and ¹¹¹In-U33 IgG blocked by i.v. pre-injection of 200 μg of cold U33IgG. Probe uptake in CWR22Rv1 xenografts is also depicted.

FIG. 9, panels A-C, show imaging of active uPA in the PC3 cardiacdissemination model with 111In-U33 IgG. Panel A depicts 2D and 3Dreconstructed ¹¹¹In-SPECT/CT images of ¹¹¹In-U33 IgG showingco-localization of the metastatic lesions with the bioluminescenceimaging (BLI) data. (top) Images showing the localization of theanti-uPA probe to an osseous metastatic lesion in the left mandible ofthe mouse (top) and probe localization to brain and lymph nodemetastatic lesions (bottom). Panel B shows inhibition of thetrypsin-like proteolytic activity by U33 IgG in the supernatant from thehomogenized mandible lesion. Supernatant from homogenate was assayed forproteolytic activity using Z-Gly-Gly-Arg-AMC (400 μM) in the presenceand absence of 100 nM U33 IgG. As shown in Panel C, the imaged brainlesion was fixed, sectioned and stained for Ki-67. Intense staining forKi-67 is apparent in the cancerous lesion (top) compared to normaladjacent brain tissue (bottom).

FIG. 10, panels A and B, show in vivo imaging of U33 IgG antibody (panelA) and Rituximab control (panel B) in the human oral squamous cellcarcinoma SAS xenograft tumor model.

FIG. 11, panel A shows the amino acid sequences (Light chain: SEQ IDNO:8; Heavy chain: SEQ ID NO:7) of the U33 light and heavy chainpolypeptides, with the CDRs underlined in the sequences and separatelypresented in the tables shown (CDRL1: SEQ ID NO:4; CDRL2: SEQ ID NO:5;CDRL3: SEQ ID NO:6; CDRH1: SEQ ID NO:1; CDRH2: SEQ ID NO:2; CDRH3: SEQID NO:3). CDRS are defined by the Kabat numbering system (Johnson et al.Nucleic Acids Research, 2000, 28: 214-218). Panel B shows the nucleicacid sequences (Light chain: SEQ ID NO:9; Heavy chain: SEQ ID NO:10)that encode the light and heavy chains of the U33 Fab.

FIG. 12, panel A schematically illustrates the different domains andother features of uPA. Here, pro-uPA (or “zymogen” uPA) is depictedbefore conversion to two-chain active uPA. Panel B shows the amino acidsequence of uPA (SEQ ID NO:11). The B chain is indicated by shading.Arrows indicate certain amino acids of interest in the S1 pocket(Asp³⁷⁰, Ser³⁷¹ and Gly³⁹⁹); the S2 pocket (His²⁷²); the S3 pocket(Leu²⁷⁰ and Ala²⁷¹); and amino acids of interest essential for catalysis(e.g., His²²⁴, Asp²⁷⁵ and/or Ser³⁷⁶) Underlined are the cysteines(Cys¹⁶⁸ and Cys²⁹⁹) involved in the disulphide bridge between the A andB chains of two-chain (active) uPA.

FIG. 13 depicts U33/uPA western-blot analysis under reducing conditions,indicating that U33 specifically recognizes the B-chain of uPA.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present disclosure relates to antibodies specific for urokinase-typeplasminogen activator (uPA). According to certain embodiments, theanti-uPA antibody specifically binds to the active form of uPA (e.g.,the antibody does not bind to the zymogen form of uPA). In certainaspects, the anti-uPA antibody that specifically binds to active uPAbinds specifically to the active form of human uPA (e.g., the antibodydoes not cross-react with active forms of uPA from non-human organisms).In certain aspects, an anti-uPA antibody of the present disclosurecompetes for specific binding to uPA with plasminogen activatorinhibitor type 1 (PAI-1), where binding of the antibody to uPA resultsin internalization of a complex that includes the antibody, uPA, andurokinase-type plasminogen activator receptor (uPAR). In certainaspects, this complex does not include PAI-1. Also provided areantibodies that specifically bind to uPA and compete for binding to uPAwith a uPA ligand, such as a synthetic peptide ligand (e.g.,p-aminobenzamidine, EGR-CMK, S-2444, and the like) of uPA. Thedisclosure also provides anti-uPA antibody conjugates and compositions(e.g., pharmaceutical formulations) comprising the antibodies and/orconjugates. Methods and kits related to the anti-uPA antibodies,conjugates, or formulations including the antibodies and/or conjugatesare also provided.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, exemplarymethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantigen” includes a plurality of such antigens and reference to “thepeptide” includes reference to one or more peptides and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

When describing the antibodies, conjugates, pharmaceutical formulationscontaining such, and methods of producing and using such antibodies,conjugates, or formulations, the following terms have the followingmeanings unless otherwise indicated. It should also be understood thatany of the moieties defined forth below may be substituted with avariety of substituents, and that the respective definitions areintended to include such substituted moieties within their scope.

The terms “polypeptide” and “protein” are used interchangeablythroughout the application and mean at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides,peptides, and fragments thereof. The protein may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures. Thus “amino acid”, or “peptide residue”, as used hereinmeans both naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. Normally, the aminoacids are in the (S) or L-configuration. If non-naturally occurring sidechains are used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation. Naturally occurring amino acidsare normally used and the protein is a cellular protein that is eitherendogenous or expressed recombinantly. The terms includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; fusion proteins withdetectable fusion partners, e.g., fusion proteins including as a fusionpartner a fluorescent protein, β-galactosidase, luciferase, etc.; andthe like. Polypeptides may be of any size, and the term “peptide” refersto polypeptides that are 5-50 residues (e.g., 8-20 residues) in length.

As used herein, the term “orientation,” refers to the positionalrelationship of a protease substrate relative the protease to which itis bound. By convention, the orientation of a substrate to its proteaseis specified from N-terminus to C-terminus based on sites named Pn, . .. , P3, P2, P1, P1′, P2′, P3′, . . . , Pn′, where P1-P1′ denotes thescissile bond to be cleaved by the protease and n is the number of thefeature relative to the scissile bond. Their respective binding sites onthe protease are named Sn, . . . , S3, S2, S1, S1′, S2′, S3′, . . . ,Sn′ and n is the number of the feature relative to the active site. Inaccordance with this nomenclature, the scissile bond of a cleavablesubstrate is presented to the active site in an N-terminus to C-terminusorientation relative to sites S1 and S1′. If the scissile bond of asubstrate is presented to the active site in a C-terminus to N-terminusorientation relative to sites S1 and S1′, the scissile bond isconsidered to be in the “reversed orientation.”

By “nucleic acid” herein is meant either DNA or RNA, or molecules whichcontain both deoxy- and ribonucleotides. Nucleic acid may be naturallyoccurring or synthetically made, and as such, includes analogs ofnaturally occurring polynucleotides in which one or more nucleotides aremodified over naturally occurring nucleotides.

The term “chemotherapy” as used herein refers to use of an agent (e.g.,drug, antibody, etc.), particularly an agent(s) that is destructive to acancerous cell, in treatment of a disease, with treatment of cancerbeing of particular interest.

A “cancer cell” as used herein refers to a cell exhibiting a neoplasticcellular phenotype, which may be characterized by one or more of, forexample, abnormal cell growth, abnormal cellular proliferation, loss ofdensity dependent growth inhibition, anchorage-independent growthpotential, ability to promote tumor growth and/or development in animmunocompromised non-human animal model, and/or any appropriateindicator of cellular transformation. “Cancer cell” may be usedinterchangeably herein with “tumor cell” or “cancerous cell”, andencompasses cancer cells of a solid tumor, a semi-solid tumor, a primarytumor, a metastatic tumor, and the like.

The term “conjugated” generally refers to a chemical linkage, eithercovalent or non-covalent, usually covalent, that proximally associatesone molecule of interest with second molecule of interest.

The terms “antigen” and “epitope” are well understood in the art andrefer to the portion of a macromolecule (e.g., a polypeptide) which isspecifically recognized by a component of the immune system, e.g., anantibody or a T-cell antigen receptor. As used herein, the term“antigen” encompasses antigenic epitopes, e.g., fragments of an antigenwhich are antigenic epitopes. Epitopes can be recognized by antibodiesin solution, e.g. free from other molecules. Epitopes can be recognizedby T-cell antigen receptor when the epitope is associated with a class Ior class II major histocompatibility complex molecule.

The terms “derivative” and “variant” refer to without limitation anycompound or antibody which has a structure or sequence derived from thecompounds and antibodies of the present disclosure and whosestructure/sequence is sufficiently similar to those disclosed herein andbased upon that similarity, would be expected, by one skilled in theart, to exhibit the same or similar activities and utilities as theclaimed and/or referenced compounds or antibody.

The term “effective amount” of a composition as provided herein isintended to mean a non-lethal but sufficient amount of the compositionto provide the desired utility. For instance, for eliciting a favorableresponse in a subject to treat a disease (e.g., cancer), the effectiveamount is the amount which eliminates or diminishes the symptomsassociated with the disorder, e.g., so as to provide for control ofcancer metastatis, to eliminate cancer cells, and/or the like. As willbe pointed out below, the exact amount required will vary from subjectto subject, depending on the species, age, and general condition of thesubject, the severity of the condition or disease that is being treated,the particular composition used, its mode of administration, and thelike. Thus, it is not possible to specify an exact “effective amount.”However, an appropriate effective amount may be determined by one ofordinary skill in the art using only routine experimentation.

The term “in combination with” as used herein refers to uses where, forexample, a first therapy is administered during the entire course ofadministration of a second therapy; where the first therapy isadministered for a period of time that is overlapping with theadministration of the second therapy, e.g. where administration of thefirst therapy begins before the administration of the second therapy andthe administration of the first therapy ends before the administrationof the second therapy ends; where the administration of the secondtherapy begins before the administration of the first therapy and theadministration of the second therapy ends before the administration ofthe first therapy ends; where the administration of the first therapybegins before administration of the second therapy begins and theadministration of the second therapy ends before the administration ofthe first therapy ends; where the administration of the second therapybegins before administration of the first therapy begins and theadministration of the first therapy ends before the administration ofthe second therapy ends. As such, “in combination” can also refer toregimen involving administration of two or more therapies. “Incombination with” as used herein also refers to administration of two ormore therapies which may be administered in the same or differentformulations, by the same or different routes, and in the same ordifferent dosage form type.

The term “isolated” is intended to mean that a compound is separatedfrom all or some of the components that accompany it in nature.“Isolated” also refers to the state of a compound separated from all orsome of the components that accompany it during manufacture (e.g.,chemical synthesis, recombinant expression, culture medium, and thelike).

The term “antibody” (also used interchangeably with “immunoglobulin”)encompasses polyclonal and monoclonal antibody preparations where theantibody may be of any class of interest (e.g., IgG, IgM, and subclassesthereof), as well as preparations including hybrid antibodies, alteredantibodies, F(ab′)₂ fragments, F(ab) molecules, Fv fragments, singlechain fragment variable displayed on phage (scFv), single chainantibodies, single domain antibodies, diabodies, chimeric antibodies,humanized antibodies, and functional fragments thereof which exhibitimmunological binding properties of the parent antibody molecule. Insome embodiments, e.g., cancer therapy, antibodies that provide forcomplement-mediated killing and/or antibody-dependent cellularcytotoxicity (ADCC) are of interest. The antibodies described herein maybe detectably labeled, e.g., with a radioisotope, an enzyme whichgenerates a detectable product, a fluorescent protein, and the like.Detectable labels that find use in in vivo imaging are of interest. Theantibodies may be further conjugated to other moieties, such as acytotoxic molecule or other molecule (e.g., to provide for delivery ofan anti-cancer drug to a cancer cell), members of specific bindingpairs, e.g., biotin (member of biotin-avidin specific binding pair), andthe like. The antibodies may also be bound to a support (e.g., a solidsupport), such as a polystyrene plate or bead, test strip, and the like.

Immunoglobulin polypeptides include the kappa and lambda light chainsand the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon and muheavy chains or equivalents in other species. Full-length immunoglobulin“light chains” (usually of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

An immunoglobulin light or heavy chain variable region is composed of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, 1991, and Lefranc et al. IMGT,the international ImMunoGeneTics information System®. Nucl. Acids Res.,2005, 33, D593-D597)). A detailed discussion of the Kabat numberingsystem is provided on the World Wide Web atkabatdatabase.com/index.html. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs. The CDRs are primarily responsible forbinding to an epitope of an antigen.

The term “monoclonal antibody” refers to an antibody composition havinga homogeneous antibody population. The term is not limited by the mannerin which it is made. The term encompasses whole immunoglobulinmolecules, as well as Fab molecules, F(ab′)2 fragments, Fv fragments,single chain fragment variable displayed on phage (scFv), fusionproteins comprising an antigen-binding portion of an antibody and anon-antibody protein, and other molecules that exhibit immunologicalbinding properties of the parent monoclonal antibody molecule. Methodsof making and screening polyclonal and monoclonal antibodies are knownin the art.

The term “specific binding of an antibody” or “antigen-specificantibody” in the context of a characteristic of an antibody refers tothe ability of an antibody to preferentially bind to a particularantigen that is present in a homogeneous mixture of different antigens.In certain embodiments, a specific binding interaction will discriminatebetween desirable and undesirable antigens (or “target” and “non-target”antigens) in a sample, in some embodiments more than about 10 to100-fold or more (e.g., more than about 1000- or 10,000-fold). Incertain embodiments, the affinity between an antibody and antigen whenthey are specifically bound in an antibody-antigen complex ischaracterized by a K_(D) (dissociation constant) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

“Conservative amino acid substitution” refers to a substitution of oneamino acid residue for another sharing chemical and physical propertiesof the amino acid side chain (e.g., charge, size,hydrophobicity/hydrophilicity). “Conservative substitutions” areintended to include substitution within the following groups of aminoacid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr. Conservative amino acid substitutions in thecontext of an antibody disclosed herein are selected so as to preservethe interaction between the antibody and the protease of interest.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material is of amedically acceptable quality and composition that may be administered toan individual along with the selected active pharmaceutical ingredientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The term “pharmaceutically acceptable carrier” as used herein refers toany suitable substance which provides a pharmaceutically acceptablevehicle for administration of a compound(s) of interest to a subject.“Pharmaceutically acceptable carrier” can encompass substances referredto as pharmaceutically acceptable diluents, pharmaceutically acceptableadditives and pharmaceutically acceptable excipients.

The term “purified” is intended to mean a compound of interest has beenseparated from components that accompany it in nature and provided in anenriched form. “Purified” also refers to a compound of interestseparated from components that can accompany it during manufacture(e.g., in chemical synthesis, recombinant expression, culture medium,and the like) and provided in an enriched form. Typically, a compound issubstantially pure when it is at least 50% to 60%, by weight, free fromorganic molecules with which it is naturally associated or with which itis associated during manufacture. Generally, the preparation is at least75%, more usually at least 90%, and generally at least 99%, by weight,of the compound of interest. A substantially pure compound can beobtained, for example, by extraction from a natural source (e.g.,bacteria), by chemically synthesizing a compound, or by a combination ofpurification and chemical modification. A substantially pure compoundcan also be obtained by, for example, enriching a sample having acompound that binds an antibody of interest. Purity can be measured byany appropriate method, e.g., chromatography, mass spectroscopy, HPLCanalysis, etc.

The term “subject” is intended to cover humans, mammals and otheranimals which contain uPA in any fashion. The terms “subject,” “host,”“patient,” and “individual” are used interchangeably herein to refer toany mammalian subject for whom diagnosis or therapy is desired,particularly humans. Other subjects may include cattle, dogs, cats,guinea pigs, rabbits, rats, mice, horses, and so on.

In the context of cancer therapies and diagnostics described herein,“subject” or “patient” is used interchangeably herein to refer to asubject having, suspected of having, or at risk of developing a tumor.The cancer may be one associated with metastasis, where the metastaticcells or cells surrounding the same (e.g., stromal cells) secretepro-uPA, which is then converted to uPA. Samples obtained from suchsubject are likewise suitable for use in the methods of the presentdisclosure.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

It is further noted that the claims may be drafted to exclude anyoptional or alternative element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed. To the extent a definitionof a term set out in a document incorporated herein by referenceconflicts with the definition of a term explicitly defined herein, thedefinition set out herein controls.

Anti-uPA antibodies are described first in greater detail, followed bydescriptions of exemplary formulations and methods employing the same,as well as a discussion of representative applications in which theantibodies, formulations and methods of the present disclosure find use.

Anti-uPA Antibodies

The present disclosure provides antibodies that specifically bind tourokinase-type plasminogen activator (uPA). The antibodies may benon-human antibodies (e.g., mouse antibodies, rabbit antibodies, or anynon-human antibody), humanized antibodies, or fully human antibodies(e.g., a fully human recombinant antibody identified from a phagedisplay library, or the like). Antibodies of the present disclosure bindto one or more epitopes of uPA. According to certain embodiments, theantibody binds to the heavy (B) chain of uPA and not the light (A) chainof uPA. Such an antibody may bind to the serine protease domain (SPD) ofuPA. An antibody that binds to the SPD of uPA may bind to the activesite of uPA, or may bind to a region outside of the active site. Incertain aspects, the anti-uPA antibody does not bind to the autolysisloop of uPA. According to certain embodiments, the anti-uPA antibodydoes not bind to the activation domain of uPA. In certain aspects, theantibody specifically binds to urokinase-type plasminogen activator(uPA) and competes for binding to uPA with a uPA ligand. The uPA ligandmay be a synthetic ligand of uPA (e.g., synthetic peptide ligand, asmall molecule ligand, or the like) or a naturally occurring ligand suchas plasminogen, fibronectin, PAI-1, uPAR, hepatocyte growth factor, orthe like).

In certain aspects, the uPA ligand is a synthetic ligand, e.g.,synthetic substrate or synthetic inhibitor). A synthetic ligand of uPAcan be a small molecule, such as p-aminobenzamidine, or a syntheticpeptide ligand (e.g., a synthetic peptide substrate or synthetic peptideinhibitor), such as synthetic peptide ligands having the amino acidsequence Glu-Gly-Arg, e.g., such as Glu-Gly-Arg-chloromethylketone(EGR-CMK), H-D-Glu-Gly-Arg-p-nitroanilide (S-2444) and the like. The uPAligand may be plasminogen, fibronectin, PAI-1, uPAR, hepatocyte growthfactor, or the like. In certain aspects, the antibody competes forbinding to uPA with a uPA ligand, which uPA ligand binds to the activesite of uPA without binding to an exosite of uPA (e.g., the ligand bindsto the active site of uPA independent of any interactions with anexosite (e.g., a region outside of the active site) of uPA). Suchexosite-independent uPA ligands may be a synthetic ligand for uPA. Theantibodies provided by the present disclosure may include one or more(e.g., 1, 2, 3, 4, 5, or all 6) complementary determining regions (CDRs)set forth in Table 1, FIG. 11 and SEQ ID NOs:1-6, or conservativevariants of one or more such CDRs.

According to certain embodiments, an anti-uPA antibody of the presentdisclosure binds to an epitope provided by the active site of uPA. Incertain aspects, the antibody that binds to the active site of uPAinteracts with one or more amino acids of uPA which are determinants forbinding a tripeptide substrate to uPA. Such an amino acid(s) includesbut is not limited to: an amino acid in the S1 pocket (e.g., Asp370,Ser371 and/or Gly399); an amino acid in the S2 pocket (e.g., His272); anamino acid in the S3 pocket (Leu270 and/or Ala271); an amino acidsessential for catalysis (e.g., His224, Asp275 and/or Ser376); and anycombination of such amino acids. Numbering of the amino acids isaccording to the uPA amino acid sequence shown in FIG. 12. Additionalinformation regarding uPA amino acids involved in substrate binding isfound, e.g., in Spraggon et al. (1995) Structure 3:681-691.

In certain aspects, provided is an antibody that competes for specificbinding to uPA with plasminogen activator inhibitor type 1 (PAI-1),where binding of the antibody to uPA results in internalization of acomplex that includes the antibody, uPA, and urokinase-type plasminogenactivator receptor (uPAR). According to certain embodiments, thiscomplex does not include PAI-1. In certain aspects, the antibody isantibody U33. The structural basis for recognition of uPA by PAI-1 hasbeen elucidated via crystallographic analysis at 2.3-angstrom resolution(Lin et al. (2011) J. Biol. Chem. 286(9):7027-7032). The crystalstructure reveals extensive interaction between the reactive center loop(RCL) of PAI-1 and catalytic sites within the active site of uPA. On theN-terminal side (P side) of the scissile bond, the P1 residue occupiesthe S1 specificity pocket of uPA, and further interacts with uPA througha well defined network of hydrogen bonds and Van der Waals interactions.The S2 pocket and S3 pocket of uPA are occupied by P2 and P3 of PAI-1,respectively. In sum, the interactions between PAI-1 and the uPA activesite region account for approximately 64% of the total contact area.

Accordingly, in certain aspects, an anti-uPA antibody of the presentdisclosure may interact with and/or shield access to one or morecatalytic sites within the active site of uPA. The anti-uPA antibody mayinteract with (e.g., occupy) one or more of the S1, S2, S3, and/or S4pockets of uPA. According to one embodiment, a portion of the anti-uPAantibody occupies and/or shields access to the S1 pocket of uPA.

According to certain embodiments, provided is an antibody thatspecifically binds to uPA and competes for binding to uPA with a uPAligand. In certain aspects, the uPA ligand is a synthetic ligand, e.g.,a small molecule such as p-aminobenzamidine, or a synthetic peptideligand, e.g., a synthetic peptide inhibitor such asGlu-Gly-Arg-chloromethylketone (EGR-CMK), a synthetic peptide substrateH-D-Glu-Gly-Arg-p-nitroanilide (S-2444) and the like, or any othersynthetic substance capable of binding to and/or being cleaved by uPA.When the synthetic ligand is a synthetic peptide ligand, the ligand mayinclude less than 20 amino acids, less than 15 amino acids, less than 10amino acids, or less than 5 amino acids (e.g., 3 amino acids). Incertain aspects, the synthetic peptide ligand is the uPA inhibitorGlu-Gly-Arg-chloromethylketone (EGR-CMK). In other aspects, thesynthetic peptide ligand is H-D-Glu-Gly-Arg-p-nitroanilide (S-2444). Incertain aspects, the antibody is the U33 Fab or an antibody having thebinding characteristics of U33 Fab (e.g., an antibody having one or more(e.g., all) of the heavy and light chain CDRs of U33 Fab). The crystalstructure of the catalytic domain of uPA complexed with EGR-CMK has beendetermined at 2.5-angstrom resolution (Spraggon et al. (1995) Structure3:681-691). This structure reveals that EGR-CMK covalently binds to theactive site of uPA to form a hemiketal structure. The C-terminalarginine of the inhibitor, EGR-CMK, is covalently linked via thecarbonyl group to Ser195 and through its methyl group to His57. Thearginine side chain of the inhibitor extends into the S1 pocket of uPAand makes a charge interaction with Asp189 at the base of the pocket.The glutamic acid of the inhibitor forms a salt bridge to Arg217 and canform a hydrogen bond to the nitrogen of Glu-218. Accordingly, theantibody that specifically binds to uPA and competes with the uPAinhibitor Glu-Gly-Arg-chloromethylketone (EGR-CMK) for binding to uPAmay interact with and/or shield access to one or more catalytic siteswithin the active site of uPA. For example, the anti-uPA antibody mayinteract with (e.g., occupy) one or more of the S1, S2, S3, and/or S4pockets of uPA. According to one embodiment, the anti-uPA antibodyoccupies and/or shields access to the S1 pocket of uPA.

In certain aspects, the antibody competes for binding to uPA with a uPAligand, which ligand binds to the active site of uPA without binding toan exosite of uPA (e.g., the ligand binds to the active site of uPAindependent of any interactions with an exosite (e.g., a region outsideof the active site of uPA).

According to certain embodiments, an anti-uPA antibody of the presentdisclosure may compete for specific binding to uPA withp-aminobenzamidine. For example, the antibody may be capable ofdisplacing p-aminobenzamidine from the S1 pocket of uPA. Such anantibody may interact with residues of the S1 pocket of uPA or one ormore regions adjacent thereto, which residues interact withp-aminobenzamidine when p-aminobenzamidine is bound to uPA.

According to certain embodiments, an anti-uPA antibody of the presentdisclosure competes for binding to uPA with a uPA ligand (e.g., PAI-1and/or a synthetic peptide ligand) and binds outside of the uPA activesite. That is, an antibody of the present disclosure may specificallybind outside of the uPA active site (e.g., a non-active site portion ofthe serine protease domain), where the binding causes a conformationalchange in uPA that prevents binding of a uPA ligand to the active siteof uPA. In some embodiments the uPA ligand is a synthetic uPA ligand.Such a conformational change may block entry of one or more portions ofthe uPA active site and prevent interactions between, e.g., PAI-1 and/orEGR-CMK with catalytic and/or non-catalytic residues within the uPAactive site.

Any of the antibodies of the present disclosure described hereinabove orbelow may be specific for the active form of uPA (e.g, do not bind tothe zymogen form of uPA). Any of the antibodies of the presentdisclosure described hereinabove or below may be specific for human uPA(e.g., do not bind to uPA from a non-human organism, such as murineuPA). Any of the antibodies of the present disclosure describedhereinabove or below may be specific for the active form of human uPA.

In one embodiment, an anti-uPA antibody of the present disclosurecomprises one or more CDRs of the amino acid sequence of the mature(i.e., missing signal sequence) heavy chain variable region (VH) of U33set out in SEQ ID NO:7, or variants thereof. In some embodiments, the VHcomprises the amino acid sequence from the beginning of the CDR1 to theend of the CDR3 of any one of the heavy chain of the foregoingantibodies.

In certain aspects, the anti-uPA antibody includes a heavy chain CDR1,CDR2 or CDR3 (HCDR1, HCDR2, HCDR3), each of which are independentlyselected from the CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:2) and CDR3 (SEQID NO:3) regions of an antibody having a heavy chain variable regioncomprising the amino acid sequence of the VH region of U33 set out inSEQ ID NO:7, a nucleic acid encoding the VH region set out in SEQ IDNO:7, or encoded by a nucleic acid molecule encoding the VH region. Itis further contemplated that an anti-uPA antibody of the presentdisclosure includes a heavy chain CDR1, CDR2 or CDR3, each of which areindependently selected from the CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:2)and CDR3 (SEQ ID NO:3) regions of an antibody having a heavy chainvariable region comprising the amino acid sequence of the VH region setout in SEQ ID NO:7. In one embodiment, the anti-uPA antibody includesthe CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:2) and CDR3 (SEQ ID NO:3)regions of an antibody having a heavy chain variable region comprisingthe amino acid sequence of the VH region of U33 set out in SEQ ID NO:7.

According to certain embodiments, the amino acid sequence of theanti-uPA antibody includes one or more CDRs of the amino acid sequenceof the mature (i.e., missing signal sequence) light chain variableregion (VL) of U33 set out in SEQ ID NO:8, or variants thereof,including CDR grafted, modified, humanized, chimeric, or HumanEngineered antibodies or any other variants described herein. In someembodiments, the VL comprises the amino acid sequence from the beginningof the CDR1 to the end of the CDR3 of the light chain of any one of theforegoing antibodies.

In certain aspects, the anti-uPA antibody includes a light chain CDR1,CDR2 or CDR3 (LCDR1, LCDR2, LCDR3), each of which are independentlyselected from the CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5) and CDR3 (SEQID NO:6) regions of an antibody having a light chain variable regioncomprising the amino acid sequence of the VL region of U33 set out inSEQ ID NO:8, a nucleic acid encoding the VL region set out in SEQ IDNO:8, or encoded by a nucleic acid molecule encoding the VL region. Itis further contemplated that an anti-uPA antibody of the presentdisclosure includes a light chain CDR1, CDR2 or CDR3, each of which areindependently selected from the CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5)and CDR3 (SEQ ID NO:6) regions of an antibody having a light chainvariable region comprising the amino acid sequence of the VL region setout in SEQ ID NO:8. In one embodiment, the anti-uPA antibody includesthe CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5) and CDR3 (SEQ ID NO:6)regions of an antibody having a light chain variable region comprisingthe amino acid sequence of the VL region of U33 set out in SEQ ID NO:8.

Antibodies of the present disclosure may have a heavy chain variableregion polypeptide having a CDR1, CDR2, and CDR3 of the amino acidsequence of SEQ ID NO:7 as defined by Kabat et al. (supra). The presentdisclosure also encompasses antibodies having a heavy chain variableregion polypeptide having a CDR1, CDR2, and CDR3 of the amino acidsequence of SEQ ID NO:7 numbered according to ImMunoGenTics (IMGT(supra)) numbering. Antibodies of the present disclosure may have alight chain variable region polypeptide having a CDR1, CDR2, and CDR3 ofthe amino acid sequence of SEQ ID NO:8 as defined by Kabat et al.(supra). Also encompassed are antibodies having a light chain variableregion polypeptide having a CDR1, CDR2, and CDR3 of the amino acidsequence of SEQ ID NO:8 numbered according to ImMunoGenTics (IMGT;supra) numbering.

In another embodiment, the antibody comprises a mature heavy chainvariable region as disclosed above, a mature light chain variable regionas disclosed above, or a mature heavy chain variable region as disclosedabove and a mature light chain variable region as disclosed above.

In certain aspects, an anti-uPA antibody of the present disclosure is amonoclonal antibody that retains any one, two, three, four, five, or sixof HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of U33, optionallyincluding one or two mutations in any of such CDR(s), e.g., aconservative or non-conservative substitution, and optionally paired asset forth in Table 1.

TABLE 1 Complementarity determining regions of U33. Heavy Chain U33HCDR1 GFTFGDYAMS (SEQ ID NO: 1) HCDR2 FIRSKAYGGTTE (SEQ ID NO: 2) HCDR3IRGANWN (SEQ ID NO: 3) Light Chain LCDR1 RSSQTLMNRNGNNFLD (SEQ ID NO: 4)LCDR2 LGSNRAP (SEQ ID NO: 5) LCDR3 MQRIEFPYT (SEQ ID NO: 6)

Also provided is a monoclonal anti-uPA antibody that retains all ofHCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:2) and HCDR3 (SEQ ID NO:3), or theheavy chain variable region of SEQ ID NO:7, optionally including one ortwo mutations in any of such CDR(s), optionally further comprising anysuitable heavy chain constant region, e.g., IgG1, IgG2, IgG3, IgG4, IgM,IgA1, IgA2, or IgE, a human sequence thereof, or a hybrid thereof.

According to certain embodiments, an antibody of the present disclosureis a monoclonal antibody that retains all of LCDR1 (SEQ ID NO:4), LCDR2(SEQ ID NO:5) and LCDR3 (SEQ ID NO:6), or the light chain variableregion of SEQ ID NO:8, optionally including one or two mutations in anyof such CDR(s), optionally further including any suitable light chainconstant region, e.g., a kappa or lambda light chain constant region, ahuman sequence thereof, or a hybrid thereof.

In certain aspects, the anti-uPA antibody includes all three heavy chainCDRs of U33, all three light chain CDRs of U33, or all six CDRs of theheavy and light chains of U33, paired as set forth in Table 1. Incertain embodiments, two heavy chain CDRs from the U33 may be combinedwith a third heavy chain CDR from a different antibody. Alternatively, aHCDR1 from the U33 Fab may be combined with a HCDR2 from a differentantibody and a HCDR3 from yet another antibody, particularly where theCDRs are highly homologous. Similarly, two light chain CDRs from U33 maybe combined with a third light chain CDR from a different antibody.Alternatively, an LCDR1 from the U33 Fab may be combined with a LCDR2from a different antibody and a LCDR3 from yet another antibody,particularly where the CDRs are highly homologous.

According to certain embodiments, the anti-uPA antibody includes apolypeptide having an amino acid sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chainvariable region set out in SEQ ID NO:7 and/or an amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the light chain variable region set out in SEQ ID NO:8.Such an antibody may further include at least one, two, three, four,five or all of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. In someembodiments, the amino acid sequence with percentage identity to theheavy chain variable region may include one, two or three of the heavychain CDRs. In other embodiments, the amino acid sequence withpercentage identity to the light chain variable region may comprise one,two, or three of the light chain CDRs.

In certain aspects, an anti-uPA antibody is provided that includes apolypeptide having an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to all three HCDRs of theheavy chain variable region set out in SEQ ID NO:7. Alternatively, oradditionally, the anti-uPA antibody may include a polypeptide having anamino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to all three LCDRs of the light chainvariable region set out in SEQ ID NO:8. In a further embodiment, anantibody of the present disclosure may include a polypeptide having anamino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to all six CDRs in the heavy chain and light chain variableregions of U33 Fab as set out in SEQ ID NOs: 7 and 8, respectively.

Antibodies of the disclosure may include one, or two or more amino acidsubstitutions in the CDR regions of the antibody (e.g., non-conservativeor conservative substitutions), e.g., as compared to U33.

According to one embodiment, the residues of the framework are altered.The heavy chain framework regions which may be altered lie within theregions surrounding the heavy chain CDR residues, and the residues ofthe light chain framework regions which may be altered lie within theregions surrounding the light chain CDR residues. An amino acid withinthe framework region may be replaced, for example, with any suitableamino acid identified in a human framework or human consensus framework.

In certain aspects, an anti-uPA antibody of the present disclosure bindsto uPA (e.g., the active form of uPA, human uPA, or the active form ofhuman uPA) with an affinity (Kd) of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less (lower meaning higher bindingaffinity). According to certain embodiments, an anti-uPA antibody of thepresent disclosure binds to uPA (e.g., the active form of uPA, humanuPA, or the active form of human uPA) with at least 2-50 fold, 10-100fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%,50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or morehigher affinity (e.g., preferentially binds to uPA) compared to bindingto any other protease, e.g., a protease selected from tPA, trypsin,chymotrypsin, elastase, hK2, PSA, KLK4, KLK7, hepsin, HGFA, MT-SP1,plasmin, thrombin, and combinations thereof.

An anti-uPA antibody of the present invention may bind to active humanuPA with at least 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold,25-fold, 50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100% or more higher affinity (e.g.,preferentially binds to active human uPA) compared to binding toinactive human uPA, or active or inactive uPA from a non-human organism.

In certain aspects, an anti-uPA antibody of the present disclosurespecifically binds to the serine protease domain of uPA, and does notbind to the Kringle or EGF-like domains of the uPA chain A. For example,the anti-uPA antibody may occupy and/or bind to an epitope within ornear the uPA active site of the serine protease domain of uPA (e.g., theS1 pocket of uPA), as described in more detail above.

Nucleic acids encoding any of the antibodies of the present inventionare also provided. For example, the nuceic acid sequences encoding theU33 heavy and light chain variable polypeptides are provided in FIG. 11,panel B, the nucleic acids may be present in an expression vector forproduction of any antibody of the present disclosure in a cell.

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental non-human (e.g., mouse) monoclonal antibodies, theycan be used for the treatment of humans with far less risk ofanaphylaxis. Accordingly, in certain aspects, an anti-uPA antibody ofthe present disclosure is chimeric or humanized.

Chimeric monoclonal antibodies, in which the variable Ig domains of anon-human (e.g., mouse) monoclonal antibody are fused to human constantIg domains, can be generated using standard procedures known in the art(See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984);and, Boulianne et al, Nature 312, 643-646, (1984)).

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”); (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”); or (3) substituting human aminoacids at positions determined to be unlikely to adversely effect eitherantigen binding or protein folding, but likely to reduce immunogenicityin a human environment (e.g., HUMAN ENGINEERING™). In the presentdisclosure, humanized antibodies may include “humanized,” “veneered,”and/or “HUMAN ENGINEERED™” antibodies. These methods are disclosed in,e.g., Jones et al., Nature 321:522 525 (1986); Morrison et al., Proc.Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536(1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol.31:169-217 (1994); Studnicka et al. U.S. Pat. No. 5,766,886; Studnickaet al., (Protein Engineering 7: 805-814, 1994; Co et al., J. Immunol.152, 2968-2976 (1994); Riechmann, et al., Nature 332:323-27 (1988); andKettleborough et al., Protein Eng. 4:773-783 (1991) each of which isincorporated herein by reference.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriatefour framework regions of human variable Ig domains. This technique(Riechmann, et al., Nature 332:323-27 (1988)), utilizes the conservedframework regions (FR1-FR4) as a scaffold to support the CDR loops whichare the primary contacts with antigen. A disadvantage of CDR grafting,however, is that it can result in a humanized antibody that has asubstantially lower binding affinity than the original mouse antibody,because amino acids of the framework regions can contribute to antigenbinding, and because amino acids of the CDR loops can influence theassociation of the two variable Ig domains. To maintain the affinity ofthe humanized monoclonal antibody, the CDR grafting technique can beimproved by choosing human framework regions that most closely resemblethe framework regions of the original mouse antibody, and bysite-directed mutagenesis of single amino acids within the framework orCDRs aided by computer modeling of the antigen binding site (e.g., Co etal., J. Immunol. 152, 2968-2976 (1994)).

Antibody Fragments

Any of the anti-uPA antibodies described elsewhere herein may be in theform of an antibody fragment. Antibody fragments comprise a portion ofan intact full length antibody and can include an antigen binding orvariable region of the intact antibody. Examples of antibody fragmentsinclude Fab; Fab′; F(ab′)2; Fv fragments; diabodies; linear antibodies;single-chain antibody molecules (e.g., scFv); multispecific antibodyfragments such as bispecfic, trispecific, etc. antibodies (e.g.,diabodies, triabodies, tetrabodies); minibody; chelating recombinantantibody; tribodies or bibodies; intrabodies; nanobodies; small modularimmunopharmaceuticals (SMIP), binding-domain immunoglobulin fusionproteins; camelized antibodies; VHH containing antibodies; and otherpolypeptides formed from antibody fragments. See, e.g., Holliger &Hudson (Nat. Biotech. 23:1126-36 (2005)).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains each with a single antigen-binding site,and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, that has two “Single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of antibody, whereinthese domains are present in a single polypeptide chain. The Fvpolypeptide can further comprise a polypeptide linker between the VH andVL domains that enables the Fv to form the desired structure for antigenbinding, resulting in a single-chain antibody (scFv), in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988). For a review of scFv see Pluckthun, in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragmentconsists of the VH and CH1 domains.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a VH domain.Diabodies are bivalent antibodies in which VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g., EP404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

Antibody Conjugates

As described in more detail elsewhere herein, according to certainembodiments, the anti-uPA antibodies of the present disclosure result ininternalization of a complex that includes the antibody, uPA, andurokinase-type plasminogen activator receptor (uPAR) (which complex mayor may not include PAI-1). Antibodies having this feature find use intherapeutic and diagnostic (e.g., in vivo imaging, etc.) applications.For example, such an antibody may be conjugated to a payload, such as atherapeutic agent (e.g., a cytotoxic payload) or labeling agent (e.g.,an in vivo imaging agent), where upon binding of the antibody to uPA andinternalization of the resulting antibody/uPA/uPAR complex, thetherapeutic or labeling agent is delivered specifically to the cytosolof target cells having surface-attached uPA/uPAR (e.g., metastatic orinvasive cancer cells (e.g., castration-resistant prostate cancercells), and/or stromal cells surrounding the same). The specificinternalization of the therapeutic or labeling agent in cells havingsurface-attached uPA/uPAR reduces unwanted exposure of non-target cellsto the therapeutic agent, and can reduce toxicity upon administration.Moreover, in imaging applications (e.g., in vivo imaging for diagnostic,prognostic, and/or any other purpose), specific internalization of alabeling agent into cells having surface-attached uPA/uPAR concentratesthe labeling agent in such cells, thereby increasing the signal-to-noiseratio and diagnostic/prognostic value of the resulting images. Moreover,the internalization feature of anti-uPA antibody conjugates of thepresent disclosure permits their administration at loweramounts/concentrations than a conjugate that includes an anti-uPAantibody that does not result in such internalization, due to thetherapeutic or labeling agent being “concentrated” in the target cellsof interest (e.g., metastatic or invasive cancer cells, and/or stromalcells surrounding the same).

Accordingly, any anti-uPA antibody described herein may be inunconjugated form, or may be conjugated directly to an agent, such as atherapeutic and/or labeling (e.g., diagnostic) agent, or may beconjugated indirectly to carrier polymers comprising such othertherapeutic or labeling agents. In some embodiments, the antibody isconjugated to a cytotoxic agent such as a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablechemotherapeutic agents include: daunomycin, doxorubicin, methotrexate,and vindesine (Rowland et al., (1986) supra). Suitable toxins include:bacterial toxins such as diphtheria toxin; plant toxins such as ricin;small molecule toxins such as geldanamycin (Mandler et al J. Natl.Cancer Inst. 92(19):1573-81 (2000); Mandler et al., Bioorg. Med. Chem.Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13.786-91 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-23 (1996)), auristatins (Doronina et al., Nat.Biotech. 21: 778-84 (2003) and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)).

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are known; for example, see (Sternberger, L. A. et al., J.Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym.62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W.J. Immunol. Meth. 13:215 (1976)).

In certain aspects, the labeling agent of an anti-uPA antibody conjugateof the present disclosure is a labeling agent that finds use in in vivoimaging, such as near-infrared (NIR) optical imaging, single-photonemission computed tomography (SPECT)/CT imaging, or the like. Labelingagents that find use in such applications include, but are not limitedto, fluorescent labels and radioisotopes, or the like. In certainaspects, the labeling agent is a multi-modal in vivo imaging agent thatpermits in vivo imaging using two or more imaging approaches (e.g., seeThorp-Greenwood and Coogan (2011) Dalton Trans. 40:6129-6143).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, and can be about 100-5000 amino acid residues. At least some ofthe amino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate. Examples of agents to which the antibody can be conjugatedinclude any of the cytotoxic, chemotherapeutic agents described herein.

Conjugated antibodies can be prepared by directly conjugating anantibody component with a therapeutic agent or labeling agent. Thegeneral procedure is analogous to the indirect method of conjugationexcept that a therapeutic or labeling agent is directly attached to anoxidized antibody component. For example, a carbohydrate moiety of anantibody can be attached to polyethyleneglycol to extend half-life.

A therapeutic or labeling agent can be attached at the hinge region of areduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Pharmaceutical Compositions

Also provided by the present disclosure are pharmaceutical compositionsthat include any of the anti-uPA antibodies described herein, or any ofthe conjugates described herein, and a pharmaceutically acceptablecarrier.

Pharmaceutical compositions of the present disclosure containing ananti-uPA antibody or conjugate of the present disclosure as an activeingredient may contain pharmaceutically acceptable carriers or additivesdepending on the route of administration. Examples of such carriers oradditives include water, a pharmaceutical acceptable organic solvent,collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinylpolymer, carboxymethylcellulose sodium, polyacrylic sodium, sodiumalginate, water-soluble dextran, carboxymethyl starch sodium, pectin,methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein,gelatin, agar, diglycerin, glycerin, propylene glycol, polyethyleneglycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serumalbumin (HSA), mannitol, sorbitol, lactose, a pharmaceuticallyacceptable surfactant and the like. Additives used are chosen from, butnot limited to, the above or combinations thereof, as appropriate,depending on the dosage form of the present disclosure.

Formulation of the pharmaceutical compositions of the present disclosurewill vary according to the route of administration selected (e.g.,solution, emulsion). An appropriate composition comprising the antibodyto be administered can be prepared in a physiologically acceptablevehicle or carrier. For solutions or emulsions, suitable carriersinclude, for example, aqueous or alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers.

A variety of aqueous carriers, e.g., sterile phosphate buffered salinesolutions, bacteriostatic water, water, buffered water, 0.4% saline,0.3% glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Therapeutic formulations of the anti-uPA antibodies of the presentdisclosure are prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The antibody or conjugate may also be entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulation herein may also contain more than one active compound(e.g., a second active agent in addition to the anti-uPA antibody orconjugate thereof) as necessary for the particular indication beingtreated (e.g., cancer), and which may be selected to complementaryactivities that do not adversely affect each other. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The anti-uPA antibodies of the present disclosure may be lyophilized forstorage and reconstituted in a suitable carrier prior to use. Thistechnique has been shown to be effective with conventionalimmunoglobulins. Any suitable lyophilization and reconstitutiontechniques can be employed. Lyophilization and reconstitution can leadto varying degrees of antibody activity loss and that use levels mayhave to be adjusted to compensate.

Methods of Production

As discussed above, the present disclosure provides antibodies thatspecifically bind to uPA. Such antibodies are highly specific forbinding to uPA. Example methods of making an anti-uPA antibody arepresented below.

Antibodies can be prepared using a wide variety of techniques known inthe art including the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, antibody may bemade and isolated using methods of phage display. The antibody may alsobe isolated from sera of an animal host immunized with an immunogeniccomposition comprising uPA, which encompasses whole proteins andfragments thereof. Exemplary antibodies include an isolated antibodycapable of specifically binding to uPA (e.g., the active form of humanuPA, where the antibody competes for specific binding to uPA with PAI-1or EGR-CMK, and/or results in internalization of a uPA/uPAR complex,which complex may not include PAI-1).

The antigen that coats the wells for phage display panning or theimmunogenic composition used to elicit the antibody of the presentdisclosure may comprise an aggregate of one or more antigens. The methodmay involve exposing antigens to an aggregating condition so as to forman aggregate. Thus the methods of production described above may furtherinclude a step of forming an aggregate of the isolated antigens.Examples of the aggregating conditions include heating, addition of anexcipient that facilitates aggregation, and the like.

Antigens used to coat the wells for phage panning or to elicitantibodies of the present disclosure may be conjugated to anothermolecule. For example, the antigen can be conjugated to a secondmolecule such as a peptide, polypeptide, lipid, carbohydrate and thelike that aids in solubility, storage or other handling properties, cellpermeability, half-life, controls release and/or distribution such as bytargeting a particular cell (e.g., neurons, leucocytes etc.) or cellularlocation (e.g., lysosome, endosome, mitochondria etc.), tissue or otherbodily location (e.g., blood, neural tissue, particular organs etc.).

A particular embodiment of of an antigen conjugated to a second moleculeis where the second molecule is an immunomodulator. “Immunomodulator” isa molecule that directly or indirectly modifies an immune response. Aspecific class of immunomodulators includes those that stimulate or aidin the stimulation of an immunological response. Examples includeantigens and antigen carriers such as a toxin or derivative thereof,including tetanus toxoid.

Phage Display

Phage display is used for the high-throughput screening of proteininteractions. Phages may be utilized to display antigen-binding domainsexpressed from a repertoire or combinatorial antibody library (e.g.,human or murine). Phage expressing an antigen binding domain that bindsthe protease of interest can be selected or identified with the proteaseof interest, e.g., using labeled uPA or uPA bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 binding domains expressed from phage withFab, Fv (individual Fv region from light or heavy chains) or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Exemplary methods are set forth, forexample, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R.and Chames, Immunol. Today 2000, 21:371; Nagy et al. Nat. Med. 2002,8:801; Huie et al., Proc. Natl. Acad. Sci. USA 2001, 98:2682; Lui etal., J. Mol. Biol. 2002, 315:1063, each of which is incorporated hereinby reference. Several publications (e.g., Marks et al., Bio/Technology1992, 10:779-783) have described the production of high affinity humanantibodies by chain shuffling, as well as combinatorial infection and invivo recombination as a strategy for constructing large phage libraries.In another embodiment, ribosomal display can be used to replacebacteriophage as the display platform (see, e.g., Hanes et al., Nat.Biotechnol. 2000, 18:1287; Wilson et al., Proc. Natl. Acad. Sci. USA2001, 98:3750; or Irving et al., J. Immunol. Methods 2001, 248:31). Cellsurface libraries may be screened for antibodies (Boder et al., Proc.Natl. Acad. Sci. USA 2000, 97:10701; Daugherty et al., J. Immunol.Methods 2000, 243:211). Such procedures provide alternatives totraditional hybridoma techniques for the isolation and subsequentcloning of monoclonal antibodies. See the Examples section below.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. For example, DNA sequences encoding heavy chain variable(VH) and light chain variable (VL) regions are amplified or otherwiseisolated from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. The DNAencoding the VH and VL regions may be joined together by an scFv linkerby PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3HSS). The vector is electroporated in E. coli and the E. coli isinfected with helper phage. The VH or VL regions are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to an antigen ofinterest (e.g., the serine protease domain of uPA) can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead.

Additional examples of phage display methods that may be used to makethe antibodies include those disclosed in PCT Application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the references listed above, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in PCT publication WO 92/22324; Mullinax et al.,BioTechniques 1992, 12:864-869; and Sawai et al., AJRI 1995, 34:26-34;and Better et al., Science 1988, 240:1041-1043 (said referencesincorporated by reference in their entireties).

Immunization and Antibody Production

The method of eliciting antibodies in a host animal involvesadministering an effective amount of uPA or a fragment thereof asantigens described above to the host animal (i.e., a suitable mammalsuch as a mouse, rabbit or guinea pig, or a suitable avian, such as achicken) to elicit production of an antibody that specifically binds touPA. Methods of immunizing animal, including the adjuvants used, boosterschedules, sites of injection, suitable animals, etc. are wellunderstood in the art, e.g., Harlow et al. (Antibodies: A LaboratoryManual, First Edition (1988) Cold spring Harbor, N.Y.), andadministration of living cells to animals has been described for severalmammals and birds, e.g., McKenzie et al (Oncogene 4:543-8, 1989),Scuderi et al (Med. Oncol. Tumor Pharmacother 2:233-42, 1985), Roth etal (Surgery 96:264-72, 1984) and Drebin et al (Nature 312:545-8, 1984).Next, a population of antibody producing cells is generated. In oneembodiment, the population of cells is produced using hybridoma methodsthat well known to one of skill in the art (see, e.g., HarlowAntibodies: A Laboratory Manual, First Edition (1988) Cold SpringHarbor, N.Y.). Cells are fused to immortalized cells, such as myelomacells or transformed cells, which are capable of replicatingindefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The immortal cell line utilized canbe selected to be deficient in enzymes necessary for the utilization ofcertain nutrients. Many such cell lines (such as myelomas) are known tothose skilled in the art, and include, for example: thymidine kinase(TK) or hypoxanthine-guanine phosphoriboxyl transferase (HGPRT). Thesedeficiencies allow selection for fused cells according to their abilityto grow on, for example, hypoxanthine aminopterinthymidine medium (HAT).In alternative embodiments, populations of cells expressing monoclonalantibodies may be made using phage display methods.

Anti-uPA antibodies, including antigen binding fragments of anti-upAantibodies, may also be produced by genetic engineering. In thistechnique, as with the standard hybridoma procedure, antibody-producingcells are sensitized to the desired antigen or immunogen. The messengerRNA isolated from the immune spleen cells or hybridomas is used as atemplate to make cDNA using PCR amplification. A library of vectors,each containing one heavy chain gene and one light chain gene retainingthe initial antigen specificity, is produced by insertion of appropriatesections of the amplified immunoglobulin cDNA into the expressionvectors. A combinatorial library can be constructed by combining theheavy chain gene library with the light chain gene library. This resultsin a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost (e.g. bacteria, insect cells, mammalian cells, or other suitableprotein production host cell.). When antibody gene synthesis is inducedin the transfected host, the heavy and light chain proteinsself-assemble to produce active antibodies that can be detected byscreening with the antigen or immunogen.

Phage Panning and Screening

Once the population of antibody-producing cells or phages is produced,the antibodies are screened using one or a combination of a variety ofassays. In general, these assays are functional assays, and may begrouped as follows: assays that detect an antibody's binding affinity orspecificity, and assays that detect the ability of an antibody toinitialize or inhibit a process.

For example, the antigen is coupled to beads or wells or other solidsupport and incubated with phage displaying the antibody of interest.After washings, bound phage is then recovered by inoculation of logphase E. coli cells. The cells are grown and expanded with helper phage.Steps are repeated for the amplification of tightly bound phages. Thephage-infected E. coli colonies after several round of enrichment areharvested and Fab antibodies are purified from the periplasmicfractions. The purified antibodies are then analyzed in accordance withmethods known in the art. Certain exemplary examples are detailed below.

The population of antibody isolated from phage-infected cells orhybridomas is further analyzed and/or screened for binding to a singleantigen (i.e., antigens that are not mixed with other antigens of theplurality of antigens) of the plurality of antigens in vitro or in situ(e.g. on cells). Immunospecific binding may be carried out according tomethods routine and known in the art. The immunoassays which can be usedinclude, but are not limited to, competitive and non-competitive assaysystems using techniques such as western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, to name but a few. See, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety.

Antibodies of the present disclosure may also be screened in vivo. Themethod involves administering an anti-uPA antibody to an animal modelfor a disease or condition and determining the effect of the antibody onthe disease or condition of the model animal. In vivo assays of theinvention include controls, where suitable controls include a sample inthe absence of the antibody. Generally, a plurality of assay mixtures isrun in parallel with different antibody concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

A monoclonal antibody of interest is one that modulates, i.e., reducesor increases a symptom of the animal model disease or condition by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 90%, ormore, when compared to a control in the absence of the antibody. Ingeneral, a monoclonal antibody of interest will cause a subject animalto be more similar to an equivalent animal that is not suffering fromthe disease or condition. Antibodies that have therapeutic value thathave been identified using the methods and compositions of the inventionare termed “therapeutic” antibodies.

Selected monoclonal antibodies of interest can be expanded in vitro,using routine tissue culture methods, or in vivo, using mammaliansubjects. For example, pristane-primed mice can be inoculated with logphase hybridoma cells in PBS for ascites production. Ascites fluid canbe stored at −70° C. prior to further purification.

Methods of Screening

A screening method provided by the present disclosure may involve theuse of a phage library to screen for an antibody that specifically bindsto uPA, and optionally having any of the additional features describedherein (competes for uPA binding with PIA-1 and/or ERG-CMK, and/orresults in internalization of a uPA/uPAR complex, which complex may notinclude PAI-1). The binding agent may be selected for its potentinhibition of uPA and/or its specific binding affinity. The method maybe executed according to the phage display method described above.

Briefly, uPA or a fragment thereof may be immobilized on an ELISA plateor on beads through a covalent or non-covalent interaction, such ashydrophobic adsorption, biotin-avidin interaction, and Ni²⁺-6×Hisinteraction. The phage library is then incubated with the immobilizedantigen/protease, washed, and recovered. During panning and selection,the bound phage is recovered and amplified in E. coli. Multiplesuccessive selection rounds ensure a selection of a phage displaying apolypeptide that acts as an antibody specific for uPA. The stringency ofthe washes increases over a number of rounds (e.g. three). Manytechniques well known in the art may be employed to increase thespecificity of the recovered phage. Examples include increased washtimes, increased detergent concentrations, increased saltconcentrations, and inclusion of known macromolecular inhibitors (e.g.,small peptidic substrates, BPTI, Ecotin, and/or previously identifiedantibody inhibitors). Identification of inhibitory antibodies mayinclude ELISAs and inhibition assays. Details on the assays to beperformed in the method for selecting and isolating an anti-uPA antibodyare discussed above.

Also contemplated by the present disclosure is a library of nucleic acidconstructs encoding the candidate anti-uPA antibodies described herein.The library encodes a plurality of candidate anti-uPA antibodies thatmay have one or more polypeptide regions in common (e.g. a heavy chainCDR3) and at least one other polypeptide region that varies among thepopulation.

Diagnostics Methods

The present disclosure provides a method of detecting uPA in vivo, or ina biological sample in situ or isolated from a subject. The methods areuseful to both diagnostic and prognostic purposes. The methods generallyinvolve contacting a sample comprising a cell with an anti-uPA antibody;and detecting binding of the antibody to a cell in the sample. The cellcan be in vivo (e.g., a tumor or stromal cell in a human subject), invitro (where the cell is in a biological sample obtained from a subjectsuspected for having cancer cells), a subject undergoing treatment,and/or a subject being tested for susceptibility to treatment.

The anti-uPA antibodies or conjugates thereof can be used to detect uPAin a biological sample of a subject having or suspected of havingcancerous cells. Such diagnostics can be useful to identify patientsamenable to the therapies disclosed herein, and/or to monitortumor/metastatic progression, response to therapy, and/or the like.

Suitable immunodiagnostic techniques include, but are not necessarilylimited to, both in vitro and in vivo (imaging) methods. The phrase “invivo imaging” as used herein refers to methods of detecting the presenceof uPA (e.g. detectably labeled U33) in a whole, live mammal. Opticallydetectable proteins such as fluorescent antibodies,radioisotope-conjugated antibodies, luciferase-conjugated antibodies,and the like may be detected by in vivo imaging. Methods for usingluciferases for real-time imaging of luciferase expression in liveanimals can be readily adapted for use in the methods disclosed herein(e.g., Greer L F et al., Luminescence 2002, 17: 43-74). In vivo imagingof fluorescent proteins in live animals is described in, e.g., Hoffman,Cell Death and Differentiation 2002, 9:786-789. See Example 13 fordetails. In vivo imaging may be used to provide 2-D as well as 3-Dimages of a mammal. Radiolabeled antibodies, for example, may beadministered to a subject and the subject imaged with a gamma camera.Charge-coupled device cameras, CMOS, or 3D tomographers may used tocarry out in vivo imaging. For example, Burdette J E Journal of Mol.Endocrin., 40: 253-261, 2008, reviews utilizing computed tomography,magnetic resonance imaging, ultrasonography, positron emissiontomography, single-photon emission computed tomography (SPECT), etc.,for in vivo imaging. SPECT can also be used with an integrated x-ray CAT(CT) scanner (SPECT/CT) in the methods. In certain aspects, thediagnostic methods utilize near-infrared (NIR) imaging. The informationfrom many in vivo imaging methods as those described above can provide3D distribution of the antibodies in the subject. See the Examplessection below for more details.

Where the methods are in vitro, the biological sample can be any samplein which uPA may be present, including but not limited to, blood samples(including whole blood, serum, etc.), tissues, whole cells (e.g., intactcells), and tissue or cell extracts. For example, the assay can involvedetection of a protease on cells in a histological tissue sample. Forexample, the tissue sample may be fixed (e.g., by formalin treatment)and may be provided embedded in a support (e.g., in paraffin) or frozenunfixed tissue.

Assays can take a wide variety of forms, such as competition, directreaction, or sandwich type assays. Exemplary assays include Westernblots; agglutination tests; enzyme-labeled and mediated immunoassays,such as enzyme-linked immunosorbent assays (ELISAs); biotin/avidin typeassays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation,and the like. The reactions generally include detectable labels such asfluorescent, chemiluminescent, radioactive, enzymatic labels or dyemolecules, or other methods for detecting the formation of a complexbetween antigen in the sample and the antibody or antibodies reactedtherewith.

The assays can involve separation of unbound antibody in a liquid phasefrom a solid phase support to which antigen-antibody complexes arebound. Solid supports which can be used include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

Where a solid support is used, the solid support is usually firstreacted with a solid phase component (e.g., an anti-uPA antibody) undersuitable binding conditions such that the component is sufficientlyimmobilized to the support. Sometimes, immobilization to the support canbe enhanced by first coupling the antibody to a protein with betterbinding properties, or that provides for immobilization of the antibodyon the support with out significant loss of antibody binding activity orspecificity. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind antibodies to asupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like, with theproviso that the molecule used to immobilize the antibody does notadversely impact the ability of the antibody to specifically bindantigen. Such molecules and methods of coupling these molecules to theantibodies, are well known to those of ordinary skill in the art. See,e.g., Brinkley, M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al.,J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu and Staros,International J. of Peptide and Protein Res. (1987) 30:117-124.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing a serin protease undersuitable binding conditions. After washing to remove any non-boundligand, a secondary binder moiety is added under suitable bindingconditions, wherein the secondary binder is capable of associatingselectively with the bound ligand. The presence or absence of thesecondary binder can then be detected using techniques well known in theart.

An ELISA method can be used, where the wells of a microtiter plate arecoated with a an anti-uPA antibody of the present disclosure. Abiological sample containing or suspected of containing a protease(e.g., a tumor cell expressing uPA), is then added to the coated wells.After a period of incubation sufficient to allow antibody binding, theplate(s) can be washed to remove unbound moieties and a detectablylabeled secondary binding molecule added. The secondary binding moleculeis allowed to react with any captured antigen, the plate washed and thepresence or absence of the secondary binding molecule detected usingmethods well known in the art.

Where desired, the presence or absence of bound uPA from a biologicalsample can be readily detected using a secondary binder comprising anantibody directed against the antibody ligands. For example, a number ofanti-bovine immunoglobulin (Ig) molecules are known in the art which canbe readily conjugated to a detectable enzyme label, such as horseradishperoxidase, alkaline phosphatase or urease, using methods known to thoseof skill in the art. An appropriate enzyme substrate is then used togenerate a detectable signal. In other related embodiments,competitive-type ELISA techniques can be practiced using methods knownto those skilled in the art.

Assays can also be conducted in solution, such that the antibodies anduPA form complexes under precipitating conditions. For example, theantibody can be attached to a solid phase particle (e.g., an agarosebead or the like) using coupling techniques known in the art, such as bydirect chemical or indirect coupling. The antibody-coated particle isthen contacted under suitable binding conditions with a biologicalsample suspected of containing uPA to provide for formation ofparticle-antibody-uPA complex aggregates which can be precipitated andseparated from the sample using washing and/or centrifugation. Thereaction mixture can be analyzed to determine the presence or absence ofantibody-antigen complexes using any of a number of standard methods,such as those immunodiagnostic methods described above.

The test sample used in the diagnostics assays can be any sample inwhich uPA may be present, including but not limited to, blood samples(including whole blood, serum, etc.), tissues, whole cells (e.g., intactcells), and tissue or cell extracts containing cells (e.g., tissue,isolated cells, etc.), a cell lysate (i.e., a sample containingnon-intact cells), where each type of sample can contain elements ofboth types (e.g., a sample of cells can contain cell lysates, and viceversa). In some embodiments, particularly as in embodiments involvingdetection of cancer cells, it may be desirable to conduct the assayusing a sample from the subject to be diagnosed that contains intact,living cells. uPA detection can then be assessed on an extracellularsurface of the cells, and can further be assessed during cell division.

Diagnostic assays can also be conducted in situ. For example, anti-uPAantibodies can be detectably labeled, administered to a subjectsuspected of having a cancer characterized by uPA activity, and bounddetectably labeled antibody detected using imaging methods available inthe art.

The diagnostic assays described herein can be used to determine whethera subject has a cancer that is more or less amenable to therapy usingantibody-based therapy, as well as monitor the progress of treatment ina subject. It also may be used to assess the course of other combinationtherapies (e.g., anti-uPA antibody therapy) as described in (U.S. Ser.No. 11/645,255 and PCT Application No. US2006/048850; incorporatedherein by reference). Thus, the diagnostic assays can inform selectionof therapy and treatment regimen by a clinician.

The protease of interest can be detected by detection of specificbinding of an antibody, e.g., a monoclonal antibody (mAb) that has theantigen-binding specificity of U33. In this embodiment, uPA may bepresent on the cell surface at any stage of the cell cycle, includingduring cell division. In some instances, cancers that present theantigen during cell division may present a lower or no detectable levelof the antigen when the cell is quiescent (i.e., not undergoing celldivision). The antigen can also be detected in a permeabilized testcell. For example, a test cancer cell that exhibits a pattern ofstaining with a U33 Fab (or an antibody having the antigen bindingspecificity of U33) that is distinct from a pattern of antibody stainingin a normal cell is identified as a cancerous cell that exhibits aU33-reactive antigen. Such cancers are thus amenable to therapy with anantibody that specifically binds the U33-reactive antigen (e.g., the mAbU33).

The above-described assay reagents, including the antibodies generatedby immunization with uPA according to the methods described previously,can be provided in kits, with suitable instructions and other necessaryreagents, in order to conduct immunoassays as described above. The kitcan also contain, depending on the particular immunoassay used, suitablelabels and other packaged reagents and materials (i.e. wash buffers andthe like). Standard immunoassays, such as those described above, can beconducted using these kits.

Methods of Treating uPA-Related Disorder

The present disclosure provides methods of treating a disorder (e.g.,cancer) related to the activity of uPA. The methods generally involveadministering to a subject in need thereof a therapeutically effectiveamount of any anti-uPA antibody or conjugate described herein, alone(e.g., in monotherapy) or in combination (e.g., in combination therapy)with one or more additional therapeutic agents. In certain aspects, themethods include treating a uPA-related disorder (e.g., cancer) byadministering to a patient in need thereof a therapeutically effectiveamount of an anti-uPA antibody (e.g., an antibody of the presentdisclosure having any of the competitive binding and/or internalizationfeatures described herein) that specifically binds to the active form ofhuman uPA, and that reduces or inhibits growth and/or invasiveness ofcells having surface-attached uPA.

By “treatment” is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition (e.g., cancer) being treated. As such, treatment also includessituations where the pathological condition, or at least symptomsassociated therewith, are completely inhibited, e.g., prevented fromhappening, or stopped, e.g. terminated, such that the host no longersuffers from the condition, or at least the symptoms that characterizethe condition. Thus treatment includes: (i) prevention, that is,reducing the risk of development of clinical symptoms, including causingthe clinical symptoms not to develop, e.g., preventing diseaseprogression to a harmful state; (ii) inhibition, that is, arresting thedevelopment or further development of clinical symptoms, e.g.,mitigating or completely inhibiting an active disease, e.g., so as todecrease tumor load, which decrease can include elimination ofdetectable cancerous cells, or so as to protect against disease causedby bacterial infection, which protection can include elimination ofdetectable bacterial cells; and/or (iii) relief, that is, causing theregression of clinical symptoms.

A variety of hosts are treatable according to the methods. Generallysuch hosts are “mammals” or “mammalian,” where these terms are usedbroadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Prodrugs of the antibody composition of the present disclosure are alsocontemplated in the methods described herein. Such prodrugs are ingeneral functional derivatives of the compounds that are readilyconvertible in vivo into the required compounds. Thus, in the methods ofthe present disclosure, the term “administering” encompassesadministering the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to the subject in needthereof. Conventional procedures for the selection and preparation ofsuitable prodrug derivatives are described, e.g., in Wermuth, “DesigningProdrugs and Bioprecursors” in Wermuth, ed. The Practice of MedicinalChemistry, 2d Ed., pp. 561-586 (Academic Press 2003). Prodrugs includeesters that hydrolyze in vivo (e.g., in the human body) to produce acompound described herein. Suitable ester groups include, withoutlimitation, those derived from pharmaceutically acceptable, aliphaticcarboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic andalkanedioic acids, in which each alkyl or alkenyl moiety has no morethan 6 carbon atoms. Illustrative esters include formates, acetates,propionates, butyrates, acrylates, citrates, succinates, andethylsuccinates.

Antibody and conjugate compositions/formulations described herein may beadministered to a subject (e.g. a human patient) to, for example, reducethe viability and/or invasiveness of cancerous cells, e.g., to reducetumor size or metastasis, reduce tumor load, and/or improve the clinicaloutcome in patients. In certain aspects, antibody compositions can beused to disrupt the cell cycle of the cancer cell, and facilitate entryof the cell into apoptosis, e.g., by inducing cancerous cells to enterthe pre-GO cell cycle phase. The methods relating to cancer contemplatedherein include, for example, use of antibody therapy alone or incombination with anti-cancer vaccine or therapy, as well as use ofantibodies generated using uPA antigens in anti-cancer vaccines (e.g.,by passive immunization) or therapies. The methods are useful in thecontext of treating or preventing a wide variety of cancers. uPA-relatedcancers that may be treated using the treatment methods of the presentdisclosure include, but are not limited to, prostate cancer (e.g.,castration-resistant prostate cancer), breast cancer, gastric cancer,colorectal cancer, esophageal cancer, renal cancer, endometrial cancer,ovarian cancer, any other uPA-related cancer, and/or combinationsthereof.

In certain embodiments, the antibody compositions may be advantageouslyused in an anti-cancer therapy, particularly where the cancerous cellspresent an active uPA on an extracellularly accessible cell surface. Oneexample is a cancer that presents a U33-reactive antigen. Cancers thatpresent a U33-reactive antigen can be identified by methods known in theart. Exemplary methods of detection and diagnosis are describedelsewhere herein.

Cancers particularly amenable to antibody therapy can be identified byexamining markers of cellular proliferation (e.g., Ki-67 antigen) and/orinvasiveness, e.g., by examining the presence/accessibility of activeuPA bound by U33 or by other anti-uPA antibodies provided by the presentdisclosure (e.g., as in an in vitro assay).

For example, the presence of an active uPA in normal human tissueappears to be transient and low abundance. It is prevalent primarily inabnormal cells, such as metastasing cancer cells of epithelial origin.Since expression of high levels of active uPA exists predominantly incancer cells, treatment with antibody compositions can be used to detectthe presence and localize cancer growth, induce cytotoxicity, and blocktumor growth. In addition, antibody compositions can be usedtherapeutically to effect/prevent adhesion and invasion of cancer cellsin other tissues.

Dosage

In the methods of the present disclosure, an effective amount ofanti-uPA antibody (or conjugate including the same) is administered to asubject in need thereof. For example, in some embodiments, the anti-uPAantibody or conjugate inhibits growth, metastasis and/or invasiveness ofa cancer cell(s) in a host when the anti-uPA antibody or conjugate isadministered in an effective amount. The amount administered variesdepending upon the goal of the administration, the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g., human, non-human primate, primate,etc.), the degree of resolution desired, the formulation of the anti-uPAantibody or conjugate, the treating clinician's assessment of themedical situation, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials. For example, the amount of the anti-uPA antibodyor conjugate employed to inhibit cancer cell growth, metastasis and/orinvasiveness is not more than about the amount that could otherwise beirreversibly toxic to the subject (i.e., maximum tolerated dose). Inother cases the amount is around or even well below the toxic threshold,but still in an immunoeffective concentration range, or even as low asthreshold dose.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the antibody or conjugate, andthus based on the disposition of the composition within the subject.This includes consideration of the route of administration as well asdosage amount, which can be adjusted for, e.g., parenteral (applied byroutes other than the digestive tract for systemic or local effects)applications. For instance, administration of a the anti-uPA antibody orconjugate is typically via injection and often intravenous,intramuscular, intratumoral, or a combination thereof.

The anti-uPA antibody or conjugate may be administered by infusion or bylocal injection, e.g. by infusion at a rate of about 50 mg/h to about400 mg/h, including about 75 mg/h to about 375 mg/h, about 100 mg/h toabout 350 mg/h, about 150 mg/h to about 350 mg/h, about 200 mg/h toabout 300 mg/h, about 225 mg/h to about 275 mg/h. Exemplary rates ofinfusion can achieve a desired therapeutic dose of, for example, about0.5 mg/m²/day to about 10 mg/m²/day, including about 1 mg/m²/day toabout 9 mg/m²/day, about 2 mg/m²/day to about 8 mg/m²/day, about 3mg/m²/day to about 7 mg/m²/day, about 4 mg/m²/day to about 6 mg/m²/day,about 4.5 mg/m²/day to about 5.5 mg/m²/day. Administration (e.g, byinfusion) can be repeated over a desired period, e.g., repeated over aperiod of about 1 day to about 5 days or once every several days, forexample, about five days, over about 1 month, about 2 months, etc. Italso can be administered prior, at the time of, or after othertherapeutic interventions, such as surgical intervention to removecancerous cells. The anti-uPA antibody or conjugate can also beadministered as part of a combination therapy, in which at least one ofan immunotherapy, a cancer chemotherapy or a radiation therapy isadministered to the subject.

Disposition of the antibody or conjugate and its correspondingbiological activity within a subject is typically gauged against thefraction of antibody present at a target of interest. For example, anantibody once administered can accumulate with a glycoconjugate or otherbiological target that concentrates the material in cancer cells andcancerous tissue. Thus dosing regimens in which the antibody isadministered so as to accumulate in a target of interest over time canbe part of a strategy to allow for lower individual doses. This can alsomean that, for example, the dose of antibody that are cleared moreslowly in vivo can be lowered relative to the effective concentrationcalculated from in vitro assays (e.g., effective amount in vitroapproximates mM concentration, versus less than mM concentrations invivo).

As an example, the effective amount of a dose or dosing regimen can begauged from the IC50 of a given antibody for inhibiting or binding uPA.By “IC50” is intended the concentration of a drug required for 50%inhibition in vitro. Alternatively, the effective amount can be gaugedfrom the EC50 of a given antibody concentration. By “EC50” is intendedthe plasma concentration required for obtaining 50% of a maximum effectin vivo.

In general, with respect to the anti-uPA antibody or conjugate of thepresent disclosure, an effective amount is usually not more than 200×the calculated IC50. Typically, the amount of an antibody or conjugatethat is administered is less than about 200×, less than about 150×, lessthen about 100× and many embodiments less than about 75×, less thanabout 60×, 50×, 45×, 40×, 35×, 30×, 25×, 20×, 15×, 10× and even lessthan about 8× or 2× than the calculated IC50. In one embodiment, theeffective amount is about 1× to 50× of the calculated IC50, andsometimes about 2× to 40×, about 3× to 30× or about 4× to 20× of thecalculated IC50. In other embodiments, the effective amount is the sameas the calculated IC50, and in certain embodiments the effective amountis an amount that is more than the calculated IC50.

An effective amount may not be more than 100× the calculated EC50. Forinstance, the amount of antibody or conjugate that is administered isless than about 100×, less than about 50×, less than about 40×, 35×,30×, or 25× and many embodiments less than about 20×, less than about15× and even less than about 10×, 9×, 9×, 7×, 6×, 5×, 4×, 3×, 2× or 1×than the calculated EC50. In one embodiment, the effective amount isabout 1× to 30× of the calculated EC50, and sometimes about 1× to 20×,or about 1× to 10× of the calculated EC50. In other embodiments, theeffective amount is the same as the calculated EC50, and in certainembodiments the effective amount is an amount that is more than thecalculated EC50.

Effective amounts can readily be determined empirically from assays,from safety and escalation and dose range trials, individualclinician-patient relationships, as well as in vitro and in vivo assayssuch as those described herein and illustrated in the Examples section,below.

The IC50 may be calculated by inhibiting antibody binding in vitro. Thisaspect can be carried out by assessing the ability of the antibody ofinterest to inhibit U33 Fab binding to uPA. In general, the procedure iscarried out by standard ELISA in which the plates are coated with uPA,e.g., at a suitable concentration, and then processed and employed todetermine inhibition of antibody binding and the IC50. These antibodiesand others suitable for various aspects of this purpose can be employed.

Routes of Administration

In practicing the methods, routes of administration (path by which theanti-uPA antibody or conjugate is brought into a subject) may vary,where representative routes of administration for the anti-uPA antibodyor conjugate are described in greater detail below. The anti-uPAantibody or conjugate alone or in combinations described above can beadministered systemically (e.g., by parenteral administration, e.g., byan intravenous route) or locally (e.g., at a local tumor site, e.g., byintratumoral administration (e.g., into a solid tumor, into an involvedlymph node in a lymphoma or leukemia), administration into a bloodvessel supplying a solid tumor, etc.).

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present disclosure calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms depend on the particular compound employed and the effectto be achieved, and the pharmacodynamics associated with each compoundin the host.

Kits & Systems

Also provided are kits and systems that find use in practicing themethods, as described above. For example, kits and systems may includeone or more of the compositions described herein, such as an anti-uPAantibody (e.g. U33), a nucleic acid encoding the same (especially anucleic acid encoding a CDR of a heavy and/or light chain of U33), or arecombinant cell containing the same. Other optional components of thekit include: buffers, etc., for administering the anti-uPA antibody,and/or for performing a diagnostic assay. The recombinant nucleic acidsof the kit may also have restrictions sites, multiple cloning sites,primer sites, etc to facilitate their ligation to constant regions ofnon-U33 encoding nucleic acids. The various components of the kit may bepresent in separate containers or certain compatible components may bepre-combined into a single container, as desired.

The kits and systems for practicing the methods may include one or morepharmaceutical formulations that include the antibody compositionsdescribed herein. As such, the kits may include a single pharmaceuticalcomposition present as one or more unit dosages. In yet otherembodiments, the kits may include two or more separate pharmaceuticalcompositions.

In addition to the above components, the kits may further includeinstructions for practicing the methods. These instructions may bepresent in the kits in a variety of forms, one or more of which may bepresent in or on the kit. One form in which these instructions may bepresent is as printed information on a suitable medium or substrate,e.g., a piece or pieces of paper on which the information is printed, inor on the packaging of the kit, in a package insert, etc. Yet anothermeans would be a computer readable medium, e.g., diskette, CD, etc., onwhich the information has been recorded. Yet another means that may bepresent is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

A kit may be provided for use in treating a host suffering from, e.g., acellular proliferative disease. This kit includes a pharmaceuticalcomposition comprising antibody specific for uPA (e.g., the active formof human uPA), and instructions for the effective use of thepharmaceutical composition in a method of treating a host suffering froma cancerous condition by inhibiting the growth of a cancer cell in asubject. Such instructions may include not only the appropriate handlingproperties, dosing regiment and method of administration, and the like,but can further include instructions to optionally screen the subjectfor an active uPA associated with the disease. This aspect can assistthe practitioner of the kit in gauging the potential responsiveness ofthe subject to treatment with an antibody of the present disclosure,including timing and duration of treatment relative to the type andgrowth stage of the cancer. Thus in another embodiment, the kit mayfurther include an antibody (e.g., U33) or other reagent for detectingan active uPA on an extracellularly accessible surface of a cancer cell.In another embodiment, the kit includes antibody that comprises aconjugate with a detectable label, such as a label suitable for in vivoimaging, e.g., a fluorophore, radionuclide, and/or the like.

The term “system” as employed herein refers to a collection ofantibodies described herein and one or more second therapeutic agents,present in single or disparate compositions that are brought togetherfor the purpose of practicing the methods. For example, separatelyobtained antibody specific to uPA and chemotherapy dosage forms broughttogether and coadministered to a subject are a system according to thepresent disclosure.

The following examples further illustrate the present invention andshould not be construed as in any way limiting its scope.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Example 1 Identification of uPA in Prostate Cancer

Quantitative PCR (qPCR) was performed to determine plasminogenactivation system (PAS) expression in prostate cancer cell lines and tocorrelate expression with androgen receptor (AR) status and cell lineaggressiveness (FIG. 1, panel A). PAS expression was observed in the ARnegative, aggressive metastatic cell lines PC3 and DU145. No expressionwas observed in AR positive prostate cancer cells lines, normal prostateepithelial cells (PrEC), or in the bladder cancer cell line TSU. mRNAexpression for uPAR and uPA were highest in PC3, while DU145 expressedsignificantly higher PAI-1. PC3 and DU145 cells were cultured in 5% O₂to mimic the O₂ deprived environment of prostate cancers and the levelsof the PAS were analyzed (FIG. 1, panel B) (Stewart et al. (2010) BJUInt 105, 8-13). After 72 hrs, hypoxia had induced a two-fold expressionincrease of the PAS members in PC3. DU145 was less affected by hypoxiawith only uPAR expression significantly increased. Under normaloxygenation conditions, PC3 expressed more uPA mRNA than DU145, and thisresult was corroborated at the protein level by IHC using a commerciallyavailable antibody (sc-14019) that recognized total uPA (zymogen uPA,active uPA and PAI-1 bound uPA) (FIG. 1, panel C). Staining in the PC3xenograft section with sc-14019 was greater than the DU145 xenograftsection for total uPA. Total uPA was visualized in prostate cancertissue microarrays using immunofluorescence (IF) (FIG. 1, panels D-G).Total uPA protein was detected in low and high grade adenocarcinomas andin osseous metastases with IF. Consistent with previous findings, uPAwas located in both the epithelium and stroma (Usher et al. (2005) Int JCancer 113, 870-880).

Example 2 U33 Fab Identification and Development

A human naïve B cell phage display library with a diversity of 4.1×10¹⁰was used to identify inhibitory antibodies against human uPA. After fourrounds of panning, 192 independent clones were screened by ELISA. Ofthese clones, 67 showed high ELISA signals and 23 had unique sequences.The 23 unique clones identified were expressed, purified, and tested foruPA inhibition. uPA inhibition was tested using Spectrozyme® uPAchromogenic substrate (American Diagnostica).

Clone U33 was identified as a Fab that exhibited inhibitory activity.U33 had a Complementarity Determining Region H3 (CDRH3) of 7 aminoacids, shorter than the CDRH3 of other inhibitory antibodies of serineproteases described in Schneider et al. (2012) J Mol Biol 415:699-715.The affinity and specificity of the U33 Fab was determined usingquantitative ELISA as well as inhibition assays with human uPA (activeand zymogen) and mouse uPA. ELISA results showed that U33 bound toactive uPA in a concentration-dependent manner, but not zymogen uPA ormouse uPA (FIG. 2, panel A). The inhibition data in FIG. 2 (panel B)indicates that U33 Fab inhibits more than 80% of human uPA activity andhas no effect on the mouse enzyme.

Under steady-state conditions U33 Fab possessed a K_(i) of 20 nM forsoluble uPA. It has been reported that the enzymatic activity of uPAbound to uPAR is considerably increased compared with the solubleprotein. U33 Fab was tested for its ability to inhibit uPAR-bound uPAusing plates coated with the uPAR-uPA complex (FIG. 2, panel C). Underthese conditions, U33 Fab appeared to be a more potent inhibitor ofreceptor-bound uPA than of soluble uPA. U33 Fab also blocked binding ofuPA to its endogenous inhibitor PAI-1 in a dose dependent manner (FIG.2, panel D). The binding of U33 Fab to uPA pre-incubated with PAI-1 wasalso tested, and U33 Fab was found to not disrupt the uPA-PAI-1 complex.U33 Fab was tested to see if it could inhibit active uPA pre-treatedwith the irreversible active site inhibitor Glu-Gly-Arg-chloromethylketone (CMK). When added to the uPA-CMK complex, the binding of U33 Fabto uPA was significantly decreased (FIG. 2, panel E). In contrast, CMKdid not inhibit binding of other anti-uPA Fabs obtained during thescreen to uPA. The conversion of U33 Fab to the full length IgGdecreased the K_(i) to 10 nM. Kinetic analysis using double reciprocalplots revealed that U33 IgG was a competitive inhibitor of uPA (FIG. 2,panel F). Additionally, U33 IgG was able to displace the non-covalentsmall molecule inhibitor p-aminobenzamidine in the active site of uPAwhen incubated with inhibited protease.

Example 3 Characterization of U33 IgG In Vitro

U33 IgG was specific for uPA when assayed against a panel of proteases.No cross reactivity was observed with proteases displaying an array ofspecificities, including the prostate cancer-associated serine proteaseshK2, PSA and KLK4 (FIG. 3, panel A). U33 IgG was tested for its abilityto inhibit trypsin-like proteolysis in PC3 and DU145 conditioned media(FIG. 3, panel B). When incubated with the generic trypsin fluorogenicsubstrate, Z-Gly-Gly-Arg-AMC, PC3 and DU145 conditioned media showedsubstantial trypsin-like activity. Addition of 100 nM U33 IgG inhibitedall trypsin-like proteolytic activity. Proteomic analysis of thesecreted proteases in the conditioned media found that both cells lineshad high levels of uPA. In PC3 conditioned media, tPA was abundant.However, the requirement of fibrin to be active eliminated tPA as asource of trypsin-like proteolytic activity Ke et al. (1997) J Biol Chem272:16603-16609. The other proteases identified in the conditioned mediawere cathepsins that either did not display activity against thesubstrate or were not active at physiologic pH.

U33 IgG labeled with ¹¹¹In via a DOTA chelate (¹¹¹In-U33 IgG) wasinternalized by PC3 cells at 37° C. with 72% of the total radioactivityinternalized within 120 minutes. A subsequent internalization studyconducted at the 120 minute time point found that ¹¹¹In-U33 IgG wasselectively internalized by cell lines expressing active uPA.Internalization was observed in DU145 cells, but was blocked in PC3 andDU145 cells pre-treated with excess cold U33 IgG. No internalizationoccurred in CWR22Rv1 cells and in PC3 cells treated with the isotypecontrol.

Western analysis under reducing conditions was performed to determinewhich chain of two-chain uPA (the A chain or the B chain) isspecifically recognized by U33. 1 μg of recombinant human uPA (R&DSystems) was run in a SDS/PAGE gel using reducing conditions andtransferred to a PVDF membrane. The membrane was blocked and incubatedwith the Fab U33 (1 μg/mL) for 2 hours. The U33 Fab was detected usingan anti-myc antibody conjugated to peroxidase. Under reducingconditions, the two chains of active human uPA run separated: B chain(approx. 34 kD) and A chain (approx. 20 kD). The results, shown in FIG.13, indicate that U33 specifically recognizes the catalytic domain (Bchain) of uPA.

ELISA assays were performed to further assess the selectivity of U33 IgGfor uPA compared to highly related serine proteases. All proteases wereactive site-titrated to ensure that identical concentrations of activeprotease were adsorbed to the ELISA plate. After blocking, increasingconcentrations of U33 IgG were incubated for 1 hour at RT. Unbound U33IgG was removed by washing and bound U33 IgG was detected with ananti-human Fc-HRP secondary. 50 μL of 2.8 nM active protease in coatingbuffer were added to each well in a Nunc Maxisorb ELISA plate andincubated O/N at 4° C. Unbound protease was removed by hand washingwells 3× with wash buffer. Wells were incubated in 200 μL of BlockSolution per well for 2 h at RT. Block Solution was removed by handwashing wells 3× with wash buffer. 50 μL of U33 IgG in Block Solutionwas added for 1 h at RT. The highest U33 IgG concentration was 651 nM,and was decreased in two-fold increments to 5.1 nM. Unbound U33 IgG wasremoved by hand washing wells 6× with wash buffer. 50 μL of 1:2000dilution of secondary antibody in Block Solution was added for 1 h atRT. Unbound secondary antibody was removed by hand washing wells 6× withwash buffer. 100 μL of 1-step Ultra TMB-ELISA was added and thenquenched with 100 μL of 1 M HCl. Signal was quantified by measuring A₄₅₀on plate reader. Results for U33 binding to uPA, thrombin, HGFA, hepsin,chymotrypsin and trypsin are shown in FIG. 4, panel A. Results for U33binding to uPA, MT-SP1 and plasmin are shown in FIG. 4, panel B. Resultsfor U33 binding to uPA and tPA are shown in FIG. 4, panel C. Results forU33 binding to human uPA and mouse uPA are shown in FIG. 4, panel D. TheELISA data show that at concentrations where U33 IgG saturates uPA,there is no detectable binding to highly related serine proteases thatinclude thrombin, HGFA, hepsin, chymotrypsin, trypsin, MT-SP1, plasmin,tPA, and mouse uPA. Therefore U33 IgG can discriminate between proteaseswith identical catalytic mechanisms and similar protein folds.

uPA is found in at least four different forms in vivo that includeinactive zymogen, active soluble uPA, active uPA bound to uPAR, andPAI-1 inhibited uPA. An ELISA assay was used to determine theselectivity of U33 IgG for these four different species. ELISA assayswere performed to further assess the selectivity of U33 IgG for activeuPA versus inactive uPA. With the following exceptions, the protocoldescribed in the previous section was utilized: human pro-uPA isinactive, therefore a MUGB titration was not performed. The proteinconcentration provided by American Diagnostica was used to prepare a 2.8nM solution of pro-uPA in PBS. Results are shown in FIG. 5, panel A.

To assess binding of U33 to uPA versus uPA complexed to uPAR, either 50μL of 2.8 nM active uPA OR 50 μL of 2.8 nM uPAR in coating buffer wasadded to each well in a Nunc Maxisorb ELISA plate and incubated O/N at4° C. Unbound protein was removed by hand washing wells 3× with washbuffer. Wells were incubated in 200 μL of Block Solution per well for 2h at RT. Block Solution was removed by hand washing wells 3× with washbuffer. 50 μL of 2.8 nM active uPA was added to uPAR coated wells andincubated at RT for 1 h. Unbound protein was removed by hand washingwells 3× with wash buffer. 50 μL of U33 IgG in Block Solution was addedfor 1 h at RT. The highest U33 IgG concentration was 651 nM, and wasdecreased in two-fold increments to 5.1 nM. All subsequent steps areidentical to the previously described protocol. Results are shown inFIG. 5, panel B.

To assess binding of U33 to uPA versus uPA inhibited by PAI-1, activeuPA was coated to an ELISA plate and wells were blocked as previouslydescribed. 50 μL of PAI-1 in Block Solution was added for 1 h at RT. Thehighest PAI-1 concentration was 133 nM, and was decreased in two-foldincrements to 8.3 nM. Unbound protein was removed by hand washing wells3× with wash buffer. 50 μL of U33 IgG in Block Solution was added for 1h at RT. The highest U33 IgG concentration was 651 nM, and was decreasedin two-fold increments to 5.1 nM. All subsequent steps are identical tothe previously described protocol. Results are shown in FIG. 5, panel C.

The above data show that U33 IgG selectively binds to all active formsof uPA but not inactive forms. Accordingly, U33 IgG is useful, e.g., asan activity-based probe.

To determine whether U33 IgG binding was dependent on an accessibleactive site, uPA was pre-treated with active site-directed inhibitorsbefore U33 IgG addition. In one experiment, uPA was pre-treated withincreasing concentration of H-Glu-Gly-Arg-CMK, a tripeptidechloromethylketone inhibitor that binds in the S3 to 51 pockets of uPAand forms a covalent bond with catalytic residues. U33 IgG binding wasassessed by ELISA. In another experiment, uPA was pre-treated withp-aminobenzamidine, a non-covalent inhibitor that binds in the S1 pocketof uPA and undergoes a change in fluorescence upon displacement.p-Aminobenzamidine displacement was measured by comparing fluorescentspectra before and after U33 IgG addition.

To assess U33 binding to uPA versus uPA-CMK, active uPA was coated to anELISA plate and wells were blocked as previously described. 50 μL ofH-Glu-Gly-Arg-CMK in Block Solution was added for 1 h at RT. The highestCMK concentration was 7.5 mM, and was decreased in two-fold incrementsto 467 μM. Unbound protein was removed by hand washing wells 3× withwash buffer. 50 μL, of U33 IgG in Block Solution was added for 1 h atRT. The highest U33 IgG concentration was 651 nM, and was decreased intwo-fold increments to 5.1 nM. All subsequent steps are identical topreviously described protocol. Results are shown in FIG. 6, panel A.

To determine whether U33 could displace p-aminobenzamidine, stocks ofuPA and p-aminobenzamidine were prepared in 1×TBST+Ca+2. Equal volumesof 100-μM p-aminobenzamidine and 2 μM uPA or buffer were combined andincubated at room temperature for 1 hour. 50 μL of thep-aminobenzamidine/uPA complex or 50 μL of p-aminobenzamidine alone wasadded per well to a blacked-walled 96 well plate. 50 μL of 12 μM U33 IgGor 50 μL of 1×TBST+Ca⁺² was added to wells containing thep-aminobenzamidine/uPA complex or p-aminobenzamidine alone. The finalconcentrations are 25-μM p-aminobenzamidine, 500-nM uPA, and 6-μM U33IgG. Fluorescence intensity measurements were made on a Tecan platereader using an excitation wavelength of 325 nM and collecting emissiondata from 356 nm-450 nm. Results are shown in FIG. 6, panel B.

The above active site binding studies indicate that U33 IgG can competewith active site directed small molecule inhibitors for uPA binding. Thedata support that U33 IgG restricts access to the S1 pocket within theuPA active site.

Example 4 U33 IgG In Vivo Imaging

U33 IgG was tested for its ability to detect active uPA in vivo usingnear-infrared (NIR) optical imaging. U33 IgG labeled with AlexaFluor 680(AF680-U33 IgG) allowed for the qualitative detection of active uPA inxenografts (FIG. 7, panel A). Maximum probe localization was achieved at72 hrs with the PC3 xenograft demonstrating high tumor uptake andretention. Less tumor uptake was observed in the DU145 xenograft inaccordance with the mRNA and IHC results. No probe localization waspresent in the CWR22Rv1 xenograft. Cryosectioning of the PC3 tumor at 72hrs, and subsequent imaging of AF680-U33 IgG with fluorescencemicroscopy, found probe penetration in the tumor tissue (FIG. 7, panelB). The accumulation of the probe in the tumor was greater than in theliver, the main clearance organ for IgG antibodies (FIG. 7, panel C).Graphing the fluorescence efficiency of the ROIs for each of the miceimaged as a function of time highlighted the uptake kinetics andselectivity of the probe.

The clinically relevant imaging modality SPECT/CT was next used toacquire three-dimensional tomographic data. ¹¹¹In-U33 IgG localizationwas seen 72 hrs post-injection in the PC3 xenograft as documented by apronounced tumor signal in the 3D data reconstruction and the 2Dtransverse view of the fused SPECT/CT image (FIG. 8, panel A). In theSPECT/CT images, noticeable hepatic clearance of the probe was observedwith little secondary accumulation in other locations. A biodistributionstudy of ¹¹¹In-U33 IgG in the PC3 xenograft found that the probeaccumulated preferentially over time in the tumor with a % ID/g of 43.2%at 72 hrs (FIG. 8, panel B). ¹¹¹In-U33 IgG had low background in vivowith a tumor-to-blood ratio of 9.9 and a tumor-to-muscle ratio of 63.Probe accumulation in the PC3 xenograft was blocked in vivo at 72 hrs bypre-treatment with excess cold U33 IgG prior to radiotracer injection.No uptake of ¹¹¹In-U33 IgG was found in the CWR22Rv1 xenograft with a %ID/g of 4.8% representing non-specific tumor localization.

¹¹¹In-U33 IgG was tested for its ability to detect small dispersedlesions that mimic human prostate cancer using a PC3 intracardiacdissemination model Park et al. (2010) Curr Protoc Pharmacol, Chapter14, Unit 14-15. The PC3 cells used for this model were engineered tostably express luciferase and the formation of experimental metastaticlesions was monitored by bioluminescence imaging (BLI) after injectionof luciferin. By week six, distinct experimental metastases had formedin the bone, brain and lymph nodes of the mice. ¹¹¹In-U33 IgG imaged apronounced osseous lesion in the jaw of this model that was identifiedby BLI (FIG. 9, panel A). In the 2D and 3D reconstructed views, thelesion (11.6 mm³ volume) was located in the left mandible. The tissue ofthis lesion was homogenized and the supernatant had marked trypsin-likeproteolytic activity, when incubated with the fluorogenic trypsinsubstrate, compared to normal control tissue extracted from the rightmandible (FIG. 9, panel B). This proteolytic activity was significantlyinhibited by the addition of 100 nM U33 IgG. In another example,¹¹¹In-U33 IgG was able to resolve a brain lesion (28.3 mm³ volume)identified by BLI (FIG. 9, panel A). Staining of the brain lession forKi-67 found that the lesion was highly proliferative compared toadjacent normal tissue (FIG. 9, panels C and D). In addition to thebrain lesion, the 2D and 3D reconstructed views also showed thedetection of lymph node lesions that were obscured by the intense signalcoming from the brain lesion (FIG. 9, panel C).

In vivo imaging of U33 IgG antibody was performed in the human oralsquamous cell carcinoma SAS xenograft tumor model. A 10-mg/kg dose ofeither AF750 labeled U33 IgG or AF750 labeled Rituximab was administeredto tumor bearing mice and images taken 72 h after administration using amethod similar to that described in the previous examples. Rituximabserves as a control for passive (i.e. antigen independent) accumulationof labeled antibody. SAS tumors do not express Rituximab's targetantigen, CD20, and Rituximab does not cross react with mouse CD20. Invivo imaging data is presented in FIG. 10 and shows greater accumulationof U33 IgG (panel A) in SAS tumors than the Rituximab control (panel B).These data suggest that U33 accumulation is due to antigen-dependentbinding in the tumor, i.e., the presence of active uPA.

A novel imaging technology for the non-invasive nuclear imaging ofaggressive cancer (e.g., prostate cancer) is described above. Thedevelopment of the SPECT imaging probe, U33 IgG, is documented from itsinitial discovery, using a human antibody identified from a Fab phagedisplay library, to its evaluation in vivo in prostate cancer models.Targeting the active form of the secreted serine protease uPA, U33 IgGis the first example of a clinically applicable cancer diagnostic thatacts by selectively inhibiting a functional enzyme secreted by cancercells. In healthy prostate tissue, the uPA promoter is epigeneticallysilenced by hypermethylation resulting in no detectable uPA in theprostate (Shukeir et al. (2006) Cancer Res 66:9202-9210; Pakneshan etal. (2003) FASEB J 17:1081-1088). As prostate cancer progresses,methylation patterns change and uPA is expressed (Pakneshan et al.(2005) Curr Cancer Drug Targets 5:471-488; Nelson et al. (2009)Endocrinology 150:3991-4002). uPA expression is epigenetically regulatedand is present regardless of AR status, making it an imaging biomarkerfor monitoring response to anti-androgens, novel therapies, and forpatient stratification. uPA expression was high in the metastatic cellslines PC3 and DU145 and expression was significantly increased in PC3cells under hypoxia. Although only two clonal derived cell linesexpressed uPA, immunofluorescence data found total uPA expression waspresent in prostate tumors of every grade and in both soft tissue andosseous metastases. These data support and further validate earlierfindings attesting to the ubiquitous nature of uPA in prostate cancer.

The development of uPA inhibitors has mainly focused on low molecularweight compounds. The further translation of these molecules has beenprevented by poor specificity and off-target effects. A previous attemptto develop an inhibitory antibody for uPA gave a human monoclonal clonalantibody with a low nanomolar affinity (Sgier et al. (2010) Protein EngDes Sel 23:261-269). This antibody could not, however, distinguishbetween active uPA and pro-uPA and lacked species specificity. Studieswith U33 Fab found the antibody could inhibit both secreted and uPARbound uPA in the low nanomolar range and was specific for the activehuman form. U33 Fab could not bind to uPA inhibited by PAI-1 or displacePAI-1 from the complex Inhibition studies against other proteases,including S1A proteases associated with prostate cancer, found U33 IgGto be a specific, competitive inhibitor of uPA. Further evidence for U33binding to the active site was provided by use of active site-directeduPA inhibitors. U33 IgG could displace a non-covalent small-moleculeinhibitor from the S1 pocket of uPA and pre-incubation of uPA with acovalent CMK inhibitor blocked U33 binding. Based on these data, thepresent inventors have demonstrated unequivocally that U33 specificallytargets active uPA in vitro and in vivo with an accuracy not seen withother activity-based probes.

The imaging properties of U33 IgG in vivo were characteristic ofantibody imaging probes that target membrane proteins. Althoughtargeting a secreted protein, U33 IgG demonstrated high tumor uptake andretention in uPA-expressing xenografts by NIR and SPECT imaging. U33 IgGwas sensitive enough to detect small osseous and soft tissue metastaticlesions a few millimeters in size using SPECT/CT. Key to the success ofU33 IgG as an imaging probe was its internalization. This occurred via atime-dependent endocytic pathway in PC3 and DU145 cells, but not in PAnegative CWR22Rv1. Internalization was blocked with excess cold U33 IgGsuggesting that internalization was due to a mechanism requiring thepresence of active uPA. Since both cell lines also express uPAR, itappears that internalization is meadiated by uPAR with U33 IgG actingthe role of PAI-1. Both PAI-1 and U33 IgG bind to the N-terminal domainof uPA, while uPAR binding occurs at the C-terminal protease domain. Invivo, this novel internalization mechanism afforded probe accumulationand sequestration of the uPA-U33 IgG complex in tumor tissue.Internalization prevented the dissemination of the uPA-U33 IgG complexto peripheral tissue resulting in high tumor uptake values thatincreased over time as demonstrated by the biodistribution. Theinternalization of U33 IgG is in direct contrast to reports thattargeted another secreted protease, prostate-specific antigen (PSA), forPET imaging using a murine IgG antibody (⁸⁹Zr-5A10) (Ulmert et al.(2012) Cancer Discov 2:320-327). With no means of internalization orbioaccumulation, ⁸⁹Zr-5A10 uptake reached its zenith 24 hrspost-injection with a low tumor-to-blood ratio.

The above data demonstrates that U33 is a novel, highly potent andselective active site inhibitor of uPA that can be used tonon-invasively image uPA related cancers (e.g., prostate cancer).Notably, the utility of U33 IgG is not limited to prostate cancer. uPAand the other components of the PAS are over-expressed and known tocontribute to the progress of a myriad of cancers (Dass et al. (2008)Cancer Treat Rev 34:122-136). U33 IgG has the potential to be both adiagnostic and therapeutic agent. The internalization and clearance fromthe blood makes U33 IgG an ideal candidate for delivering therapeuticpayloads as a drug conjugate or for radioimmunotherapy with beta andalpha emitting radionuclides. In summary, the platform technologydescribed here has the immediate potential to change the way cancer istreated, imaged, and/or the like, leading to better therapeutic optionsand prolonged patient survival.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A conjugate comprising: a monoclonal antibodycomprising: a heavy chain complementary determining region 1 (HCDR1)having the amino acid sequence of SEQ ID NO: 1; a heavy chaincomplementary determining region 2 (HCDR2) having the amino acidsequence of SEQ ID NO: 2; a heavy chain complementary determining region3 (HCDR3) having the amino acid sequence of SEQ ID NO: 3; a light chaincomplementary determining region 1 (LCDR1) having the amino acidsequence of SEQ ID NO: 4; a light chain complementary determining region2 (LCDR2) having the amino acid sequence of SEQ ID NO: 5; and a lightchain complementary determining region 3 (LCDR3) having the amino acidsequence of SEQ ID NO: 6; and a therapeutic agent or a labeling agent.2. The conjugate of claim 1, wherein the monoclonal antibody comprises aheavy chain polypeptide comprising a variable region comprising theamino acid sequence set forth in SEQ ID NO:
 7. 3. The conjugate of claim1, wherein the monoclonal antibody comprises a light chain polypeptidecomprising a variable region comprising the amino acid sequence setforth in SEQ ID NO:
 8. 4. The conjugate of claim 1, wherein themonoclonal antibody comprises heavy and light chain polypeptidescomprising variable regions comprising the amino acid sequences setforth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
 5. The conjugateof claim 1, wherein the monoclonal antibody is selected from the groupconsisting of: an IgG, Fv, scFv, Fab, F(ab′)₂, and Fab′.
 6. Theconjugate of claim 1, wherein the agent is a therapeutic agent.
 7. Theconjugate of claim 6, wherein the therapeutic agent is a cytotoxic agentselected from the group consisting of: a radionuclide, achemotherapeutic agent, and a toxin.
 8. The conjugate of claim 1,wherein the agent is a labeling agent.
 9. The conjugate of claim 8,wherein the labeling agent is an in vivo imaging agent.
 10. A sterilepharmaceutical composition comprising: a monoclonal antibody comprising:a heavy chain complementary determining region 1 (HCDR1) having theamino acid sequence of SEQ ID NO: 1; a heavy chain complementarydetermining region 2 (HCDR2) having the amino acid sequence of SEQ IDNO: 2; a heavy chain complementary determining region 3 (HCDR3) havingthe amino acid sequence of SEQ ID NO: 3; a light chain complementarydetermining region 1 (LCDR1) having the amino acid sequence of SEQ IDNO: 4; a light chain complementary determining region 2 (LCDR2) havingthe amino acid sequence of SEQ ID NO: 5; and a light chain complementarydetermining region 3 (LCDR3) having the amino acid sequence of SEQ IDNO: 6; and a pharmaceutically acceptable carrier.
 11. The composition ofclaim 10, wherein the monoclonal antibody comprises a heavy chainpolypeptide comprising a variable region comprising the amino acidsequence set forth in SEQ ID NO:
 7. 12. The composition of claim 10,wherein the monoclonal antibody comprises a light chain polypeptidecomprising a variable region comprising the amino acid sequence setforth in SEQ ID NO:
 8. 13. The composition of claim 10, wherein themonoclonal antibody comprises heavy and light chain polypeptidescomprising variable regions comprising the amino acid sequences setforth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
 14. Thecomposition of claim 10, wherein the monoclonal antibody is selectedfrom the group consisting of: an IgG, Fv, scFv, Fab, F(ab′)₂, and Fab′.15. A method for treating cancer comprising: administering to a subjectin need thereof a therapeutically effective amount of a conjugate ofclaim 1, wherein the conjugate comprises a therapeutic agent.
 16. Themethod of claim 15, wherein the therapeutic agent is a cytotoxic agent.17. The method of claim 15, wherein the cancer is selected from thegroup consisting of: prostate cancer, breast cancer, gastric cancer,colorectal cancer, esophageal cancer, renal cancer, endometrial cancer,and ovarian cancer.
 18. The method of claim 15, wherein the cancer iscastration-resistant prostate cancer (CRPC).
 19. A method for treatingcancer comprising: administering to a subject in need thereof atherapeutically effective amount of a composition of claim
 10. 20. Themethod of claim 19, wherein the cancer is selected from the groupconsisting of: prostate cancer, breast cancer, gastric cancer,colorectal cancer, esophageal cancer, renal cancer, endometrial cancer,and ovarian cancer.
 21. The method of claim 19, wherein the cancer iscastration-resistant prostate cancer (CRPC).
 22. A method of detecting acell expressing urokinase-type plasminogen activator (uPA), the methodcomprising: contacting a cell with the conjugate of claim 1, wherein theconjugate comprises a labeling agent; and detecting binding of themonoclonal antibody of the conjugate to a cell in the sample.
 23. Themethod of claim 22, wherein the cell is suspected of being a cancerouscell.
 24. A method of detecting a cell expressing urokinase-typeplasminogen activator (uPA) in a subject, the method comprising:administering the conjugate of claim 1 to a subject, wherein theconjugate comprises a labeling agent; and detecting binding of themonoclonal antibody of the conjugate to a cell in the subject.
 25. Themethod of claim 24, wherein the subject is suspected of having cancer.26. A method of detecting a cell expressing urokinase-type plasminogenactivator (uPA), the method comprising: contacting a cell with thepharmaceutical composition of claim 10; and detecting binding of themonoclonal antibody of the pharmaceutical composition to a cell in thesample.
 27. The method of claim 26, wherein the cell is suspected ofbeing a cancerous cell.